Process for the identification of a compound which inhibits the binding of the second bromodomain of each of human BRD-2, BRD-3, and BRD-4

ABSTRACT

A process for the identification of compounds with a molecular weight in the range 100 to 750 which inhibit the binding of the first and/or second bromodomains of human BRD-2 to 4 to acetylated lysine residues of their physiological partner proteins which comprises selecting those compounds which are able to:
         a) form a hydrogen bonding interaction in which the compound accepts a hydrogen bond from the sidechain NH2 group of the asparagine residue found at:       

                                           BRD-2   BRD-2   BRD-3       BRD-4   BRD-4     BD1   BD2   BD1   BRD-3 BD2   BD1   BD2           ASN156   ASN429   ASN116   ASN391   ASN140   ASN433                              
or
         b) accept a water-mediated hydrogen bond in which the compound accepts a hydrogen bond from a water that is itself hydrogen-bonded to the sidechain hydroxyl of the tyrosine residue found at       

                                           BRD-2       BRD-3       BRD-4         BD1   BRD-2 BD2   BD1   BRD-3 BD2   BD1   BRD-4 BD2           TYR113   TYR386   TYR73   TYR348   TYR97   TYR390                              
and
         c) which are also able to form a Van der Waals interaction with a lipophilic binding region of a binding pocket such that one or more heavy atoms of the said compounds lie within a 5 Å range of any of the heavy atoms of the following bromodomain residues which define the binding pocket:       

                                           BRD-2   BRD-2   BRD-3       BRD-4   BRD-4     BD1   BD2   BD1   BRD-3 BD2   BD1   BD2           TRP97   TRP370   TRP57   TRP332   TRP81   TRP374     PRO98   PRO371   PRO58   PRO333   PRO82   PRO375     ASP161   ASP434   ASP121   GLU396   ASP145   GLU438     ILE162   VAL435   ILE122   VAL397   ILE146   VAL439     MET165   MET438   MET125   MET400   MET149   MET442                                  
pharmaceutical compositions containing such compounds, and their use in therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed pursuant to 35 USC 371 as a United StatesNational Phase Application of International Patent Application SerialNo. PCT/EP2010/066714 filed on Nov. 3, 2010, which claims priority from0919430.9 filed on Nov. 5, 2009 and 1002974.2 filed on Feb. 22, 2010 inthe United Kingdom.

FIELD OF THE INVENTION

The present invention relates to a process for the identification ofsmall molecules which inhibit the binding of the first and secondbromodomains (BD1 and 2, also known as the N- and C-terminalbromodomains) of the human BET family proteins BRD-2 to 4 to acetylatedlysine residues of their physiological partner proteins, pharmaceuticalcompositions containing such compounds and to their use in therapy.

BACKGROUND OF THE INVENTION

The genomes of eukaryotic organisms are highly organised within thenucleus of the cell. The long strands of duplex DNA are wrapped aroundan octomer of histone proteins (most usually comprising two copies ofhistones H2A, H2B H3 and H4) to form a nucleosome. This basic unit isthen further compressed by the aggregation and folding of nucleosomes toform a highly condensed chromatin structure. A range of different statesof condensation are possible, and the tightness of this structure variesduring the cell cycle, being most compact during the process of celldivision. Chromatin structure plays a critical role in regulating genetranscription, which cannot occur efficiently from highly condensedchromatin. The chromatin structure is controlled by a series of posttranslational modifications to histone proteins, notably histones H3 andH4, and most commonly within the histone tails which extend beyond thecore nucleosome structure. These modifications include acetylation,methylation, phosphorylation, ubiquitinylation, SUMOylation. Theseepigenetic marks are written and erased by specific enzymes, which placethe tags on specific residues within the histone tail, thereby formingan epigenetic code, which is then interpreted by the cell to allow genespecific regulation of chromatin structure and thereby transcription.

Histone acetylation is most usually associated with the activation ofgene transcription, as the modification loosens the interaction of theDNA and the histone octomer by changing the electrostatics. In additionto this physical change, specific proteins bind to acetylated lysineresidues within histones to read the epigenetic code. Bromodomains aresmall (˜110 amino acid) distinct domains within proteins that bind toacetylated lysine residues commonly but not exclusively in the contextof histones. There are a family of around 50 proteins known to containbromodomains, and they have a range of functions within the cell.

The BET family of bromodomain containing proteins comprises 4 proteins(BRD-2, BRD-3, BRD-4 and BRD-t) which contain tandem bromodomains (BD1and 2) capable of binding to two acetylated lysine residues in closeproximity, increasing the specificity of the interaction. BRD-2 andBRD-3 are reported to associate with histones along actively transcribedgenes and may be involved in facilitating transcriptional elongation(Leroy et al, Mol. Cell. 2008 30(1):51-60), while BRD-4 appears to beinvolved in the recruitment of the pTEF-B complex to inducible genes,resulting in phosphorylation of RNA polymerase and increasedtranscriptional output (Hargreaves et al, Cell, 2009 138(1): 129-145).It has also been reported that BRD4 or BRD3 may fuse with NUT (nuclearprotein in testis) forming novel fusion oncogenes, BRD4-NUT or BRD3-NUT,in a highly malignant form of epithelial neoplasia (French et al. CancerResearch, 2003, 63, 304-307 and French et al. Journal of ClinicalOncology, 2004, 22 (20), 4135-4139). Data suggests that BRD-NUT fusionproteins contribute to carcinogensesis (Oncogene, 2008, 27, 2237-2242).BRD-t is uniquely expressed in the testes and ovary. All family membershave been reported to have some function in controlling or executingaspects of the cell cycle, and have been shown to remain in complex withchromosomes during cell division—suggesting a role in the maintenance ofepigenetic memory. In addition some viruses make use of these proteinsto tether their genomes to the host cell chromatin, as part of theprocess of viral replication (You et al Cell, 2004 117(3):349-60).

Umehara et al have solved the X-ray crystal structure for human BRD-2BD1 when bound to a histone acetylated lysine residue (Proteincrystallographic databank entry 2dvq) and demonstrated that theacetylated lysine residue accepts a hydrogen bond from the sidechain NH2group of ASN 156 and also accepts a hydrogen bond from a water moleculethat is itself hydrogen-bonded to the sidechain hydroxyl of TYR113. Theyhave also predicted the amino acid residues which define the acetyllysine recognition pocket of the first bromodomain (BD1) of human BRD-2(JP2008-156311, The Institute of Physical and Chemical Research(RIKEN)). We have now identified small molecules which inhibit thebinding of BD1 and 2 of the human BET family proteins BRD-2 to 4 toacetylated lysine residues of their physiological partner proteins.X-ray crystal studies of these molecules when bound to these BETbromodomains have allowed us to retrospectively identify the key bindingsites involved in this interaction. This information can be used in therational drug design of further small molecules which are able toinhibit the binding of the first and/or second bromodomains of humanBRD-2 to 4 to acetylated lysine residues of their physiological partnerproteins.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a processfor the identification of small molecules, in particular compounds witha molecular weight in the range 100 to 750, which inhibit the binding ofthe first and/or second bromodomains of human BRD-2 to 4 to acetylatedlysine residues of their physiological partner proteins which comprisesselecting those compounds which are able to:

-   -   a) form a hydrogen bonding interaction in which the compound        accepts a hydrogen bond from the sidechain NH2 group of the        asparagine residue found at:

BRD-2 BRD-2 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BRD-3 BD2 BD1 BD2 ASN156ASN429 ASN116 ASN391 ASN140 ASN433or

-   -   b) accept a water-mediated hydrogen bond in which the compound        accepts a hydrogen bond from a water that is itself        hydrogen-bonded to the sidechain hydroxyl of the tyrosine        residue found at

BRD-2 BRD-3 BRD-4 BD1 BRD-2 BD2 BD1 BRD-3 BD2 BD1 BRD-4 BD2 TYR113TYR386 TYR73 TYR348 TYR97 TYR390and

-   -   c) which are also able to form a Van der Waals interaction with        a lipophilic binding region of a binding pocket such that one or        more heavy atoms of the said compounds lie within a 5 Å range of        any of the heavy atoms of the following bromodomain residues        which define the binding pocket:

BRD-2 BRD-2 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BRD-3 BD2 BD1 BD2 TRP97 TRP370TRP57 TRP332 TRP81 TRP374 PRO98 PRO371 PRO58 PRO333 PRO82 PRO375 ASP161ASP434 ASP121 GLU396 ASP145 GLU438 ILE162 VAL435 ILE122 VAL397 ILE146VAL439 MET165 MET438 MET125 MET400 MET149 MET442

From a comparison of the amino acid sequences of the human BET familybromodomains (FIG. 1) a person skilled in the art will appreciate thatthe residues shown in Table 1 are equivalent. This may also be seen bycomparison of the published crystal structures of the BRD-2, BRD-3 andBRD-4 bromodomains, which have all been solved. See Nakamura et al. (J.Biol. Chem. 2007, 282, 4193-4201) for a description of the BRD-2 D1bromodomain structure, and also protein crystallographic databankentries for BRD-2 D1 (1x0j, 2cvq, 2drv, 2dvs, 2dvq), BRD-2 D2 (2dw,2e3k), BRD-3 D1 (2nxb), BRD-3 D2 (2oo1), BRD-4 D1 (2oss) and BRD-4 D2(2ouo, 2dww).

TABLE 1 BRD-2 BD1 BRD-2 BD2 BRD-3 BD1 BRD-3 BD2 BRD-4 BD1 BRD-4 BD2 aTRP97 TRP370 TRP57 TRP332 TRP81 TRP374 b PRO98 PRO371 PRO58 PRO333 PRO82PRO375 c TYR113 TYR386 TYR73 TYR348 TYR97 TYR390 d ASN156 ASN429 ASN116ASN391 ASN140 ASN433 e ASP161 ASP434 ASP121 GLU396 ASP145 GLU438 fILE162 VAL435 ILE122 VAL397 ILE146 VAL439 g MET165 MET438 MET125 MET400MET149 MET442

In a second aspect of the present invention, there is provided apharmaceutical composition comprising a compound identified according tothe above process, or a pharmaceutically acceptable salt or solvatethereof, and one or more pharmaceutically acceptable carriers, diluentsand excipients.

In a third aspect of the present invention, there is provided a compoundidentified according to the above process, or a pharmaceuticallyacceptable salt or solvate thereof, for use in therapy, in particular inthe treatment of diseases or conditions for which a bromodomaininhibitor is indicated.

In a fourth aspect of the present invention, there is provided a methodof treating diseases or conditions for which a bromodomain inhibitor isindicated in a subject in need thereof which comprises administering atherapeutically effective amount of a compound identified according tothe above process, or a pharmaceutically acceptable salt or solvatethereof.

In a fifth aspect of the present invention, there is provided the use ofa compound identified according to the above process, or apharmaceutically acceptable salt or solvate thereof, in the manufactureof a medicament for the treatment of diseases or conditions for which abromodomain inhibitor is indicated.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Sequence alignment of the human BRD-2, BRD-3 and BRD-4bromodomains. The bromodomains have been extracted from the sequencesand aligned to each other. The first residue in each line is numberedaccording to the entry in the Swissprot sequence database (entriesP25440, BRD-2_HUMAN; Q15059, BRD-3_HUMAN; O60885, BRD-4_HUMAN). Keyresidues are labelled below: a=Trp97, b=Pro98, c=Tyr113, d=Asn156,e=Asp161, f=Ile162, g=Met165 (BRD-2 D1 residues and numbering). Asshown, position a is a conserved tryptophan; position b is a conservedproline; position c is a conserved tyrosine; position d is a conservedasparagine; position e is aspartic acid or glutamic acid; position f isisoleucine or valine, and position g is a conserved methionine.

-   SEQ ID NO: 1 is BRD-2_D1-   SEQ ID NO: 2 is BRD-3_D1-   SEQ ID NO: 3 is BRD-4_D1-   SEQ ID NO: 4 is BRD-2_D2-   SEQ ID NO: 5 is BRD-3_D2-   SEQ ID NO: 6 is BRD-4_D2

FIG. 2: Depiction of compound (I) when bound to BRD-2 BD1.

FIG. 3: Depiction of compound (I) when bound to BRD-4 BD1.

FIG. 4: Depiction of compound (I) when bound to BRD-4 BD2.

FIG. 5: Depiction of compound (II) when bound to BRD-2 BD1.

FIG. 6: Depiction of compound (II) when bound to BRD-2 BD2.

FIG. 7: Overlay of X-ray crystal structures of compounds (I) (depictedas ball+stick) and (II) (depicted as stick) when bound to BRD-2 BD1showing the compounds accepting a hydrogen bond from the sidechain NH2of ASN156 and forming a Van der Waals interaction with a lipophilicregion within the binding pocket formed by the residues TRP97, PRO98,ASP161, ILE 162 and MET165 (indicated by a dotted circle).

FIG. 8: Overlay of X-ray crystal structures of compound (I) (depicted asball+stick) when bound to BRD-4 BD2 and compound (II) (depicted asstick) when bound to BRD-2 BD2 showing the compounds accepting ahydrogen bond from a water molecule that was itself hydrogen-bonded tothe sidechain hydroxyl of the tyrosine residue found at position 386 ofBRD-2 BD2 or the equivalent 390 position of BRD-4 BD2, and forming a Vander Waals interaction with a lipophilic region within the binding pocketformed by the residues TRP370, PRO371, ASP434, VAL435 and MET438 inBRD-2 BD2 or the equivalent residues TRP374, PRO375, GLU438, VAL439 andMET442 in BRD-4 BD2 (indicated by the dotted circle).

FIG. 9: Depiction of compound (III) when bound to BRD-2 BD1.

FIG. 10: Depiction of compound (IV) when bound to BRD-2 BD1.

FIG. 11: Overlay of X-ray crystal structures of compounds (II) (depictedas ball+stick) and (IV) (depicted as stick) when bound to BRD-2 BD1showing the compounds accepting a hydrogen bond from the sidechain NH2of ASN156 and forming a Van der Waals interaction with a lipophilicregion within the binding pocket formed by the residues TRP97, PRO98,ASP161, ILE 162 and MET165 (indicated by a dotted circle).

DETAILED DESCRIPTION OF THE INVENTION

A small group of molecules which were shown by classical pharmacologicaltechniques to have an interesting but unexplained anti-inflammatorybiological profile were shown through subsequent chemoproetomic studiesto bind to the bromodomain regions of the human BET family proteins.

Thus, novel compounds which belong to two different structural classeshave been identified which inhibit the binding of the first and/orsecond bromodomains of human BRD-2 to 4 to acetylated lysine residues oftheir physiological partner proteins (hereinafter referred to asbromodomain inhibitors). Examples of these bromodomain inhibitorsinclude:

When assessed in a Fluorescence Anisotropy Binding Assay both compounds(I) and (II) above demonstrated a pIC50≧6.0 in each of the BRD-2, BRD-3and BRD-4 assays.

Analysis of X-ray crystal structures of these bromodomain inhibitorswhen bound to these BET bromodomains have allowed us to identify the keybinding sites involved in this interaction.

In particular. X-ray crystal structures were obtained for compound (I)when bound to BRD-2 BD1, BRD-4 BD1 and BRD-4 BD2, and for compound (II)when bound to BRD-2 BD1 and 2. These are given in each of FIGS. 2 to 6below.

Comparison of these crystal structures indicated that the structurallyunrelated compounds (I) and (II) interact with the key acetylated lysinebinding pocket of BRD-2 and 4 in the same way, mimicking thehydrogen-bonding network normally made by the acetylated lysine moietyof histone peptides within this pocket. One interaction was with asidechain NH2 group of an asparagine residue. Compounds (I) and (II)also interacted with a sidechain hydroxyl of a tyrosine residue via anintermediate water molecule. For example, both compounds (I) and (II)accepted a hydrogen bond from the sidechain NH2 group of the asparagineresidue found at the 156 position in BRD-2 BD 1 (see FIGS. 2, 5 and 7).Both compounds (I) and (II) also accepted a hydrogen bond from a watermolecule that was itself hydrogen-bonded to the sidechain hydroxyl ofthe tyrosine residue found at the 390 position of BRD-4 BD2 and theequivalent 386 position of BRD-2 BD2 (see FIGS. 4, 6 and 8).

Further comparison between the different crystal structures obtained ledto the identification of a third conserved interaction between thestructurally unrelated compounds (I) and (II) and the human proteinsBRD-2 and 4. Compounds (I) and (II) were each found to interact with afurther binding pocket formed partly by residues of the ZA loop of thebromodomain protein, and partly by residues found at the N-terminal endof the αC helix (see Nakamura et al, J. Biol. Chem. 2007, 282, 4193-4201for definitions). The identity of these residues in each bromodomain areas listed in Table 2.

TABLE 2 Residues corresponding to the binding pocket BRD-2 BRD-2 BRD-3BRD-4 BRD-4 BD1 BD2 BD1 BRD-3 BD2 BD1 BD2 TRP97 TRP370 TRP57 TRP332TRP81 TRP374 PRO98 PRO371 PRO58 PRO333 PRO82 PRO375 ASP161 ASP434 ASP121GLU396 ASP145 GLU438 ILE162 VAL435 ILE122 VAL397 ILE146 VAL439 MET165MET438 MET125 MET400 MET149 MET442

For example, both compounds (I) and (II) formed a Van der Waalsinteraction with a lipophilic region within this binding pocket whereinone or more heavy atoms of the compound lay within a 5 Å range of any ofthe heavy atoms of the bromodomain residues TRP97, PRO98, ASP161, ILE162 or MET165 of BRD-2 BD1 (see FIG. 7).

In particular, both compounds (I) and (II) formed a Van der Waalsinteraction with a lipophilic region within this binding pocket whereinone or more heavy atoms of the compound lay within 7.5 Å of at least oneheavy atom of each of PRO98, ASP161 and ILE162 of BRD-2 BD1 (see FIG.7).

It was noteworthy that this interaction was not observed in the crystalstructure of the histone acetylated lysine residue when bound to humanBRD-2 BD1. However, surprisingly, interaction with this further bindingpocket appears to be of particular importance in conferring activity.Compound (III) set out below shows a marked reduction in pIC50 whenassessed in the Fluorescence Anisotropy Binding Assay for the binding ofBRD-2 to 4 when compared with compound (II).

When assessed in a Fluorescence Anisotropy Binding Assay compound (III)above demonstrated a pIC50≦4.3 in each of the BRD-2, BRD-3 and BRD-4assays. % Inhibition at 200 μM compound concentration of 34%, 46% and45% was seen in the BRD-2 to 4 assays respectively, indicating thatbinding was occurring but at a low level. This is in contrast tocompound (II) which demonstrated a pIC50≧6.5 in each of the BRD-2, BRD-3and BRD-4 assays.

The crystal structure of compound (III) bound to BRD-2 BD1 indicatesthat the compound accepts a hydrogen bond from the sidechain NH2 groupof the asparagine residue found at the 156 position in BRD-2 BD 1.Compound (III) also accepts a hydrogen bond from a water that is itselfhydrogen-bonded to the sidechain hydroxyl of the tyrosine residue foundat the 113 position in BRD-2 BD1. However, there is no interaction withthe binding pocket containing the residues tryptophan, proline,asparagine, isoleucine and and methionine (see FIG. 9). Addition of thependant 4-chloroaniline group, as seen in compound (II), allowsinteraction with the binding pocket, in addition to the acetylatedlysine binding pocket as shown in FIGS. 5 and 7.

Compound (IV) is a further novel bromodomain inhibitor, which whenassessed in a Fluorescence Anisotropy Binding Assay demonstrated apIC50 >6.0 in each of the BRD-2, BRD-3 and BRD-4 assays.

The crystal structure of compound (IV) when bound to BRD-2 BD1 indicatesthat, like each of the compounds (I) to (III), the compound accepts ahydrogen bond from the sidechain NH2 group of the asparagine residuefound at the 156 position in BRD-2 BD1 and also accepts a hydrogen bondfrom a water that is itself hydrogen-bonded to the sidechain hydroxyl ofthe tyrosine residue found at the 113 position in BRD-2 BD1 (see FIG.10).

Further comparison of the X-ray crystal structures of compounds (II) and(IV) when bound to BRD-2 BD1 shows that the key Van der Waalsinteraction with a lipophilic region within the binding pocket definedby the residues set out in Table 2 was conserved even though the4-chloroaniline substituent had been replaced with an isopropylcarbamate moiety (see FIG. 11).

From a comparison of the amino acid sequences of the human BET familybromodomains (FIG. 1) a person skilled in the art will appreciate thatthe residues shown in Table 1 are equivalent. This may also be seen bycomparison of the published crystal structures of the BRD-2, BRD-3 andBRD-4 bromodomains, which have all been solved. See Nakamura et al. (J.Biol. Chem. 2007, 282, 4193-4201) for a description of the BRD-2 D1bromodomain structure, and also protein crystallographic databankentries for BRD-2 D1 (1x0j, 2cvq, 2drv, 2dvs), BRD-2 D2 (2dvv, 2e3k),BRD-3 D1 (2nxb), BRD-3 D2 (2oo1), BRD-4 D1 (2oss) and BRD-4 D2 (2ouo,2dww).

TABLE 1 BRD-2 BD1 BRD-2 BD2 BRD-3 BD1 BRD-3 BD2 BRD-4 BD1 BRD-4 BD2 aTRP97 TRP370 TRP57 TRP332 TRP81 TRP374 b PRO98 PRO371 PRO58 PRO333 PRO82PRO375 c TYR113 TYR386 TYR73 TYR348 TYR97 TYR390 d ASN156 ASN429 ASN116ASN391 ASN140 ASN433 e ASP161 ASP434 ASP121 GLU396 ASP145 GLU438 fILE162 VAL435 ILE122 VAL397 ILE146 VAL439 g MET165 MET438 MET125 MET400MET149 MET442

Thus, in a first aspect the invention provides a process for theidentification of compounds with a molecular weight in the range 100 to750 which inhibit the binding of the first and/or second bromodomains ofhuman BRD-2 to 4 to acetylated lysine residues of their physiologicalpartner proteins which comprises selecting those compounds which areable to:

-   -   a) form a hydrogen bonding interaction in which the compound        accepts a hydrogen bond from the sidechain NH2 group of the        asparagine residue found at:

BRD-2 BRD-2 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BRD-3 BD2 BD1 BD2 ASN156ASN429 ASN116 ASN391 ASN140 ASN433or

-   -   b) accept a water-mediated hydrogen bond in which the compound        accepts a hydrogen bond from a water that is itself        hydrogen-bonded to the sidechain hydroxyl of the tyrosine        residue found at

BRD-2 BRD-3 BRD-4 BD1 BRD-2 BD2 BD1 BRD-3 BD2 BD1 BRD-4 BD2 TYR113TYR386 TYR73 TYR348 TYR97 TYR390and

-   -   c) which are also able to form a Van der Waals interaction with        a lipophilic binding region of a binding pocket such that one or        more heavy atoms of the said compounds lie within a 5 Å range of        any of the heavy atoms of the following bromodomain residues        which define the binding pocket:

BRD-2 BRD-2 BRD-3 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BD2 BD1 BD2 TRP97 TRP370TRP57 TRP332 TRP81 TRP374 PRO98 PRO371 PRO58 PRO333 PRO82 PRO375 ASP161ASP434 ASP121 GLU396 ASP145 GLU438 ILE162 VAL435 ILE122 VAL397 ILE146VAL439 MET165 MET438 MET125 MET400 MET149 MET442

In a further aspect of the invention there is provided a process for theidentification of compounds with a molecular weight in the range 100 to750 which inhibit the binding of the first and/or second bromodomains ofhuman BRD-2 to 4 to acetylated lysine residues of their physiologicalpartner proteins which comprises selecting those compounds which areable to:

-   -   a) form a hydrogen bonding interaction in which the compound        accepts a hydrogen bond from the sidechain NH2 group of the        asparagine residue found at:

BRD-2 BRD-2 BRD-3 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BD2 BD1 BD2 ASN156ASN429 ASN116 ASN391 ASN140 ASN433and

-   -   b) accept a water-mediated hydrogen bond in which the compound        accepts a hydrogen bond from a water that is itself        hydrogen-bonded to the sidechain hydroxyl of the tyrosine        residue found at

BRD-2 BRD-2 BRD-3 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BD2 BD1 BD2 TYR113TYR386 TYR73 TYR348 TYR97 TYR390and

-   -   c) which are also able to form a Van der Waals interaction with        a lipophilic binding region of a binding pocket such that one or        more heavy atoms of the said compounds lie within a 5 Å range of        any of the heavy atoms of the following bromodomain residues        which define the binding pocket:

BRD-2 BRD-2 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BRD-3 BD2 BD1 BD2 TRP97 TRP370TRP57 TRP332 TRP81 TRP374 PRO98 PRO371 PRO58 PRO333 PRO82 PRO375 ASP161ASP434 ASP121 GLU396 ASP145 GLU438 ILE162 VAL435 ILE122 VAL397 ILE146VAL439 MET165 MET438 MET125 MET400 MET149 MET442

In a further aspect of the invention there is provided a process whereinstep (c) requires the compounds to be able to form a Van der Waalsinteraction with a lipophilic binding region of a binding pocket suchthat one or more heavy atoms of the said compounds lie within 7.5 Å ofat least one heavy atom of each of the 3 residues listed for a givenbromodomain

BRD-2 BRD-3 BRD-4 BD1 BRD-2 BD2 BD1 BRD-3 BD2 BD1 BRD-4 BD2 PRO98 PRO371PRO58 PRO333 PRO82 PRO375 ASP161 ASP434 ASP121 GLU396 ASP145 GLU438ILE162 VAL435 ILE122 VAL397 ILE146 VAL439

In a yet further aspect of the invention there is provided a processwherein step (a) and/or (b) is performed first to allow identificationof a compound fragment, before step (c) is performed to modify thefragment identified from steps (a) and/or (b) to provide a compound witha molecular weight in the range 100 to 750 which inhibit the binding ofthe first and/or second bromodomains of human BRD-2 to 4 to acetylatedlysine residues of their physiological partner proteins. The personskilled in the art will recognise this to be fragment based compoundidentification and optimisation.

In a further aspect the invention provides a process for theidentification of compounds with a molecular weight in the range 100 to500 which inhibit the binding of the first and/or second bromodomains ofhuman BRD-2 to 4 to acetylated lysine residues of their physiologicalpartner proteins which comprises selecting those compounds which areable to:

-   -   a) form a hydrogen bonding interaction in which the compound        accepts a hydrogen bond from the sidechain NH2 group of the        asparagine residue found at:

BRD-2 BRD-2 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BRD-3 BD2 BD1 BD2 ASN156ASN429 ASN116 ASN391 ASN140 ASN433

-   -   b) accept a water-mediated hydrogen bond in which the compound        accepts a hydrogen bond from a water that is itself        hydrogen-bonded to the sidechain hydroxyl of the tyrosine        residue found at

BRD-2 BRD-3 BRD-4 BD1 BRD-2 BD2 BD1 BRD-3 BD2 BD1 BRD-4 BD2 TYR113TYR386 TYR73 TYR348 TYR97 TYR390and

-   -   c) which are also able to form a Van der Waals interaction with        a lipophilic binding region of a binding pocket such that one or        more heavy atoms of the said compounds lie within a 5 Å range of        any of the heavy atoms of the following bromodomain residues        which define the binding pocket:

BRD-2 BRD-2 BRD-3 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BD2 BD1 BD2 TRP97 TRP370TRP57 TRP332 TRP81 TRP374 PRO98 PRO371 PRO58 PRO333 PRO82 PRO375 ASP161ASP434 ASP121 GLU396 ASP145 GLU438 ILE162 VAL435 ILE122 VAL397 ILE146VAL439 MET165 MET438 MET125 MET400 MET149 MET442

In a further aspect of the invention there is provided a process for theidentification of compounds with a molecular weight in the range 100 to500 which inhibit the binding of the first and/or second bromodomains ofhuman BRD-2 to 4 to acetylated lysine residues of their physiologicalpartner proteins which comprises selecting those compounds which areable to:

-   -   a) form a hydrogen bonding interaction in which the compound        accepts a hydrogen bond from the sidechain NH2 group of the        asparagine residue found at:

BRD-2 BRD-2 BRD-3 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BD2 BD1 BD2 ASN156ASN429 ASN116 ASN391 ASN140 ASN433and

-   -   b) accept a water-mediated hydrogen bond in which the compound        accepts a hydrogen bond from a water that is itself        hydrogen-bonded to the sidechain hydroxyl of the tyrosine        residue found at

BRD-2 BRD-2 BRD-3 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BD2 BD1 BD2 TYR113TYR386 TYR73 TYR348 TYR97 TYR390and

-   -   c) which are also able to form a Van der Waals interaction with        a lipophilic binding region of a binding pocket such that one or        more heavy atoms of the said compounds lie within a 5 Å range of        any of the heavy atoms of the following bromodomain residues        which define the binding pocket:

BRD-2 BRD-2 BRD-3 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BD2 BD1 BD2 TRP97 TRP370TRP57 TRP332 TRP81 TRP374 PRO98 PRO371 PRO58 PRO333 PRO82 PRO375 ASP161ASP434 ASP121 GLU396 ASP145 GLU438 ILE162 VAL435 ILE122 VAL397 ILE146VAL439 MET165 MET438 MET125 MET400 MET149 MET442

In a further aspect of the invention there is provided a process whereinstep (c) requires the compounds to be able to form a Van der Waalsinteraction with a lipophilic binding region of a binding pocket suchthat one or more heavy atoms of the said compounds lie within 7.5 Å ofat least one heavy atom of each of the 3 residues listed for a givenbromodomain

BRD-2 BRD-2 BRD-3 BRD-3 BRD-4 BRD-4 BD1 BD2 BD1 BD2 BD1 BD2 PRO98 PRO371PRO58 PRO333 PRO82 PRO375 ASP161 ASP434 ASP121 GLU396 ASP145 GLU438ILE162 VAL435 ILE122 VAL397 ILE146 VAL439

In a yet further aspect of the invention there is provided a processwherein step (a) and/or (b) is performed first to allow identificationof a compound fragment, before step (c) is performed to modify thefragment identified from steps (a) and/or (b) to provide a compound witha molecular weight in the range 100 to 500 which inhibit the binding ofthe first and/or second bromodomains of human BRD-2 to 4 to acetylatedlysine residues of their physiological partner proteins. The personskilled in the art will recognise this to be fragment based compoundidentification and optimisation.

There are many ways in which compounds that take advantage of theinteractions described above may be discovered or designed. In a processknown as virtual screening, molecules can be identified from databasesof real or virtual compounds. Methods to do this may make use of theprotein structure, (e.g. docking), of the 3D ligand structure, (e.g.pharmacophore searching, shape-based or field-based similaritysearching), or of the 2D ligand structure (e.g. similarity orsubstructure searching), or by combinations of these approaches. Similarmethods may also be used to design new compounds, either from firstprinciples or by modification of existing active molecules, in a processknown as de novo design. A person skilled in the art will be aware ofmany ways in which such activities can be carried out, including but notlimited to those described in review articles, recent examples of whichinclude Muegge & Oloff, Drug Discovery Today: Technologies, 2006, 3,405-411; Kontoyianni et al. Current Medicinal Chemistry, 2008, 15,107-116; Seifert & Lang, Mini-Reviews in Medicinal Chemistry, 2007,63-72; the sections of Comprehensive Medicinal Chemistry II, Vol 4:Computer-Assisted Drug Design, ed. Taylor & Triggle, Elsevier 2007.

The compounds identified using the above-mentioned processes form afurther aspect of the invention and are hereinafter referred to as“compounds of the invention”.

It will be appreciated that, whilst the compounds of the invention maybind to each of BD1 and 2 of the human BRD-2 to 4 proteins, the kineticsand binding affinity may be different at each of these binding sites.

It will be appreciated that when synthesised the compounds of theinvention may exist as a free base or a salt or solvate thereof, forexample as a pharmaceutically acceptable salt thereof. The presentinvention covers compounds of the invention as the free base and assalts thereof, for example as a pharmaceutically acceptable saltthereof. In one embodiment the invention relates to compounds of theinvention or a pharmaceutically acceptable salt thereof.

Because of their potential use in medicine, salts of the compounds ofthe invention are desirably pharmaceutically acceptable. Suitablepharmaceutically acceptable salts can include acid or base additionsalts. As used herein, the term ‘pharmaceutically acceptable salt’ meansany pharmaceutically acceptable salt or solvate of a compound of theinvention, which upon administration to the recipient is capable ofproviding (directly or indirectly). For a review on suitable salts seeBerge et al., J. Pharm. Sci., 66:1-19, (1977). Typically, apharmaceutically acceptable salt may be readily prepared by using adesired acid or base as appropriate. The resultant salt may precipitatefrom solution and be collected by filtration or may be recovered byevaporation of the solvent.

A pharmaceutically acceptable base addition salt can be formed byreaction of a compound of the invention with a suitable inorganic ororganic base, (e.g. triethylamine, ethanolamine, triethanolamine,choline, arginine, lysine or histidine), optionally in a suitablesolvent, to give the base addition salt which is usually isolated, forexample, by crystallisation and filtration. Pharmaceutically acceptablebase salts include ammonium salts, alkali metal salts such as those ofsodium and potassium, alkaline earth metal salts such as those ofcalcium and magnesium and salts with organic bases, including salts ofprimary, secondary and tertiary amines, such as isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexyl amine andN-methyl-D-glucamine.

A pharmaceutically acceptable acid addition salt can be formed byreaction of a compound of the invention with a suitable inorganic ororganic acid (such as hydrobromic, hydrochloric, sulphuric, nitric,phosphoric, succinc, maleic, acetic, propionic, fumaric, citric,tartaric, lactic, benzoic, salicylic, glutamaic, aspartic,p-toluenesulfonic, benzenesulfonic, methanesulfonic, ethanesulfonic,naphthalenesulfonic such as 2-naphthalenesulfonic, or hexanoic acid),optionally in a suitable solvent such as an organic solvent, to give thesalt which is usually isolated for example by crystallisation andfiltration. A pharmaceutically acceptable acid addition salt of acompound of the invention can comprise or be for example a hydrobromide,hydrochloride, sulfate, nitrate, phosphate, succinate, maleate, acetate,propionate, fumarate, citrate, tartrate, lactate, benzoate, salicylate,glutamate, aspartate, p-toluenesulfonate, benzenesulfonate,methanesulfonate, ethanesulfonate, naphthalenesulfonate (e.g.2-naphthalenesulfonate) or hexanoate salt.

Other non-pharmaceutically acceptable salts, e.g. formates, oxalates ortrifluoroacetates, may be used, for example in the isolation of thecompounds of the invention, and are included within the scope of thisinvention.

The invention includes within its scope all possible stoichiometric andnon-stoichiometric forms of the salts of the compounds of the invention.

It will be appreciated that many organic compounds can form complexeswith solvents in which they are reacted or from which they areprecipitated or crystallized. These complexes are known as “solvates”.For example, a complex with water is known as a “hydrate”. Solvents withhigh boiling points and/or capable of forming hydrogen bonds such aswater, xylene, N-methyl pyrrolidinone, methanol and ethanol may be usedto form solvates. Methods for identification of solvates include, butare not limited to, NMR and microanalysis. Solvates of the compounds ofthe invention are within the scope of the invention.

The invention includes within its scope all possible stoichiometric andnon-stoichiometric forms of the solvates of the compounds of theinvention.

The invention encompasses all prodrugs, of the compound of theinvention, which upon administration to the recipient is capable ofproviding (directly or indirectly) the compound of the invention, or anactive metabolite or residue thereof. Such derivatives are recognizableto those skilled in the art, without undue experimentation.Nevertheless, reference is made to the teaching of Burger's MedicinalChemistry and Drug Discovery, 5^(th) Edition, Vol 1: Principles andPractice, which is incorporated herein by reference to the extent ofteaching such derivatives.

The compounds of the invention may be in crystalline or amorphous form.Furthermore, some of the crystalline forms of the compounds of theinvention may exist as polymorphs, which are included within the scopeof the present invention. Polymorphic forms of compounds of theinvention may be characterized and differentiated using a number ofconventional analytical techniques, including, but not limited to, X-raypowder diffraction (XRPD) patterns, infrared (IR) spectra, Ramanspectra, differential scanning calorimetry (DSC), thermogravimetricanalysis (TGA) and solid state nuclear magnetic resonance (SSNMR).

Certain compounds of the invention may exist in one of severaltautomeric forms. It will be understood that the present inventionencompasses all tautomers of the compounds of the invention whether asindividual tautomers or as mixtures thereof.

It will be appreciated from the foregoing that included within the scopeof the invention are solvates, hydrates, complexes and polymorphic formsof the compounds of the invention and salts thereof.

The compounds of the invention are bromodomain inhibitors, and thus tobelieved to have potential utility in the treatment of diseases orconditions for which a bromodomain is indicated.

The present invention thus provides a compound of the invention for usein therapy. The compound of the invention can be for use in thetreatment of diseases or conditions for which a bromodomain inhibitorindicated.

The present invention thus provides a compound of the invention for usein the treatment of any diseases or conditions for which a bromodomainis indicated.

Also provided is the use of a compound of the invention in themanufacture of a medicament for the treatment of diseases or conditionsfor which a bromodomain inhibitor is indicated.

Also provided is a method of treating diseases or conditions for which abromodomain inhibitor is indicated in a subject in need thereof whichcomprises administering a therapeutically effective amount of compoundof the invention or a pharmaceutically acceptable salt thereof.

Suitably the subject in need thereof is a mammal, particularly a human.

As used herein, the term “effective amount” means that amount of a drugor pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system, animal or human that is being sought, forinstance, by a researcher or clinician. Furthermore, the term“therapeutically effective amount” means any amount which, as comparedto a corresponding subject who has not received such amount, results inimproved treatment, healing, prevention, or amelioration of a disease,disorder, or side effect, or a decrease in the rate of advancement of adisease or disorder. The term also includes within its scope amountseffective to enhance normal physiological function.

Bromodomain inhibitors are believed to be useful in the treatment of avariety of diseases or conditions related to systemic or tissueinflammation, inflammatory responses to infection or hypoxia, cellularactivation and proliferation, lipid metabolism, fibrosis and in theprevention and treatment of viral infections.

Bromodomain inhibitors may be useful in the treatment of a wide varietyof chronic autoimmune and inflammatory conditions such as rheumatoidarthritis, osteoarthritis, acute gout, psoriasis, systemic lupuserythematosus, multiple sclerosis, inflammatory bowel disease (Crohn'sdisease and Ulcerative colitis), asthma, chronic obstructive airwaysdisease, pneumonitis, myocarditis, pericarditis, myositis, eczema,dermatitis, alopecia, vitiligo, bullous skin diseases, nephritis,vasculitis, atherosclerosis, Alzheimer's disease, depression, retinitis,uveitis, scleritis, hepatitis, pancreatitis, primary biliary cirrhosis,sclerosing cholangitis, Addison's disease, hypophysitis, thyroiditis,type I diabetes and acute rejection of transplanted organs.

Bromodomain inhibitors may be useful in the treatment of a wide varietyof acute inflammatory conditions such as acute gout, giant cellarteritis, nephritis including lupus nephritis, vasculitis with organinvolvement such as glomerulonephritis, vasculitis including giant cellarteritis, Wegener's granulomatosis, Polyarteritis nodosa, Behcet'sdisease, Kawasaki disease, Takayasu's Arteritis, vasculitis with organinvolvement, acute rejection of transplanted organs.

Bromodomain inhibitors may be useful in the prevention or treatment ofdiseases or conditions which involve inflammatory responses toinfections with bacteria, viruses, fungi, parasites or their toxins,such as sepsis, sepsis syndrome, septic shock, endotoxaemia, systemicinflammatory response syndrome (SIRS), multi-organ dysfunction syndrome,toxic shock syndrome, acute lung injury, ARDS (adult respiratorydistress syndrome), acute renal failure, fulminant hepatitis, burns,acute pancreatitis, post-surgical syndromes, sarcoidosis, Herxheimerreactions, encephalitis, myelitis, meningitis, malaria and SIRSassociated with viral infections such as influenza, herpes zoster,herpes simplex and coronavirus.

Bromodomain inhibitors may be useful in the prevention or treatment ofconditions associated with ischaemia-reperfusion injury such asmyocardial infarction, cerebrovascular ischaemia (stroke), acutecoronary syndromes, renal reperfusion injury, organ transplantation,coronary artery bypass grafting, cardio-pulmonary bypass procedures,pulmonary, renal, hepatic, gastro-intestinal or peripheral limbembolism.

Bromodomain inhibitors may be useful in the treatment of disorders oflipid metabolism via the regulation of APO-A1 such ashypercholesterolemia, atherosclerosis and Alzheimer's disease.

Bromodomain inhibitors may be useful in the treatment of fibroticconditions such as idiopathic pulmonary fibrosis, renal fibrosis,post-operative stricture, keloid formation, scleroderma and cardiacfibrosis.

Bromodomain inhibitors may be useful in the prevention and treatment ofviral infections such as herpes virus, human papilloma virus, adenovirusand poxvirus and other DNA viruses.

Bromodomain inhibitors may be useful in the treatment of cancer,including hematological, epithelial including lung, breast and coloncarcinomas, midline carcinomas, mesenchymal, hepatic, renal andneurological tumours.

The term “diseases or conditions for which a bromodomain inhibitor isindicated”, is intended to include any of or all of the above diseasestates.

In one embodiment the disease or condition for which a bromodomaininhibitor is indicated is selected from diseases associated withsystemic inflammatory response syndrome, such as sepsis, burns,pancreatitis, major trauma, haemorrhage and ischaemia. In thisembodiment the bromodomain inhibitor would be administered at the pointof diagnosis to reduce the incidence of: SIRS, the onset of shock,multi-organ dysfunction syndrome, which includes the onset of acute lunginjury, ARDS, acute renal, hepatic, cardiac and gastro-intestinal injuryand mortality. In another embodiment the bromodomain inhibitor would beadministered prior to surgical or other procedures associated with ahigh risk of sepsis, haemorrhage, extensive tissue damage, SIRS or MODS(multiple organ dysfunction syndrome). In a particular embodiment thedisease or condition for which a bromodomain inhibitor is indicated issepsis, sepsis syndrome, septic shock or endotoxaemia. In anotherembodiment, the bromodomain inhibitor is indicated for the treatment ofacute or chronic pancreatitis. In another embodiment the bromodomain isindicated for the treatment of burns.

In one embodiment the disease or condition for which a bromodomaininhibitor is indicated is selected from herpes simplex infections andreactivations, cold sores, herpes zoster infections and reactivations,chickenpox, shingles, human papilloma virus, cervical neoplasia,adenovirus infections, including acute respiratory disease, poxvirusinfections such as cowpox and smallpox and African swine fever virus. Inone particular embodiment a bromodomain inhibitor is indicated for thetreatment of Human papilloma virus infections of skin or cervicalepithelia.

While it is possible that for use in therapy, a compound of theinvention as well as pharmaceutically acceptable salts thereof may beadministered as the raw chemical, it is common to present the activeingredient as a pharmaceutical composition.

The present invention therefore provides in a further aspect apharmaceutical composition comprising a compound of the invention or apharmaceutically acceptable salt and one or more or pharmaceuticallyacceptable carriers, diluents and/or excipients. The compounds of theformula (I) and pharmaceutically acceptable salts, are as describedabove. The carrier(s), diluent(s) or excipient(s) must be acceptable inthe sense of being compatible with the other ingredients of thecomposition and not deleterious to the recipient thereof. In accordancewith another aspect of the invention there is also provided a processfor the preparation of a pharmaceutical composition including admixing acompound of the formula (I), or a pharmaceutically acceptable saltthereof, with one or more pharmaceutically acceptable carriers, diluentsor excipients. The pharmaceutical composition can be for use in thetreatment of any of the conditions described herein.

Since the compounds of the invention are intended for use inpharmaceutical compositions it will be readily understood that they areeach preferably provided in substantially pure form, for example, atleast 60% pure, more suitably at least 75% pure and preferably at least85% pure, especially at least 98% pure (% in a weight for weight basis).

Pharmaceutical compositions may be presented in unit dose formscontaining a predetermined amount of active ingredient per unit dose.Preferred unit dosage compositions are those containing a daily dose orsub-dose, or an appropriate fraction thereof, of an active ingredient.Such unit doses may therefore be administered more than once a day.Preferred unit dosage compositions are those containing a daily dose orsub-dose (for administration more than once a day), as herein aboverecited, or an appropriate fraction thereof, of an active ingredient.

Pharmaceutical compositions may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, inhaled, intranasal, topical (including buccal,sublingual or transdermal), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous or intradermal) route. Suchcompositions may be prepared by any method known in the art of pharmacy,for example by bringing into association the active ingredient with thecarrier(s) or excipient(s).

In one embodiment the pharmaceutical composition is adapted forparenteral administration, particularly intravenous administration.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe composition isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Powders suitable for incorporating intotablets or capsules may be prepared by reducing the compound to asuitable fine size (e.g. by micronisation) and mixing with a similarlyprepared pharmaceutical carrier such as an edible carbohydrate, as, forexample, starch or mannitol. Flavoring, preservative, dispersing andcoloring agent can also be present.

Capsules may be made by preparing a powder mixture, as described above,and filling formed gelatin sheaths. Glidants and lubricants such ascolloidal silica, talc, magnesium stearate, calcium stearate or solidpolyethylene glycol can be added to the powder mixture before thefilling operation. A disintegrating or solubilizing agent such asagar-agar, calcium carbonate or sodium carbonate can also be added toimprove the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, glidants,lubricants, sweetening agents, flavours, disintegrating agents andcoloring agents can also be incorporated into the mixture. Suitablebinders include starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride and the like.

Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum and the like. Tablets are formulated, forexample, by preparing a powder mixture, granulating or slugging, addinga lubricant and disintegrant and pressing into tablets. A powder mixtureis prepared by mixing the compound, suitably comminuted, with a diluentor base as described above, and optionally, with a binder such ascarboxymethylcellulose, an aliginate, gelatin, or polyvinyl pyrrolidone,a solution retardant such as paraffin, a resorption accelerator such asa quaternary salt and/or an absorption agent such as bentonite, kaolinor dicalcium phosphate. The powder mixture can be granulated by wettingwith a binder such as syrup, starch paste, acadia mucilage or solutionsof cellulosic or polymeric materials and forcing through a screen. As analternative to granulating, the powder mixture can be run through thetablet machine and the result is imperfectly formed slugs broken intogranules. The granules can be lubricated to prevent sticking to thetablet forming dies by means of the addition of stearic acid, a stearatesalt, talc or mineral oil. The lubricated mixture is then compressedinto tablets. The compounds of the present invention can also becombined with a free flowing inert carrier and compressed into tabletsdirectly without going through the granulating or slugging steps. Aclear or opaque protective coating consisting of a sealing coat ofshellac, a coating of sugar or polymeric material and a polish coatingof wax can be provided. Dyestuffs can be added to these coatings todistinguish different unit dosages.

Oral fluids such as solution, syrups and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of the compound. Syrups can be prepared by dissolving thecompound in a suitably flavored aqueous solution, while elixirs areprepared through the use of a non-toxic alcoholic vehicle. Suspensionscan be formulated by dispersing the compound in a non-toxic vehicle.Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols andpolyoxy ethylene sorbitol ethers, preservatives, flavor additive such aspeppermint oil or natural sweeteners or saccharin or other artificialsweeteners, and the like can also be added.

Where appropriate, dosage unit compositions for oral administration canbe microencapsulated. The formulation can also be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax or the like.

The compounds of the invention can also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils.

For treatments of the eye or other external tissues, for example mouthand skin, the compositions are preferably applied as a topical ointmentor cream. When formulated in an ointment, the active ingredient may beemployed with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredient may be formulated in a cream withan oil-in-water cream base or a water-in-oil base.

Pharmaceutical compositions adapted for topical administrations to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent.

Dosage forms for nasal or inhaled administration may conveniently beformulated as aerosols, solutions, suspensions, gels or dry powders.

For compositions suitable and/or adapted for inhaled administration, itis preferred that the compound of the invention is in aparticle-size-reduced form e.g. obtained by micronisation. Thepreferable particle size of the size-reduced (e.g. micronised) compoundor salt is defined by a D50 value of about 0.5 to about 10 microns (forexample as measured using laser diffraction).

Aerosol formulations, e.g. for inhaled administration, can comprise asolution or fine suspension of the active substance in apharmaceutically acceptable aqueous or non-aqueous solvent. Aerosolformulations can be presented in single or multidose quantities insterile form in a sealed container, which can take the form of acartridge or refill for use with an atomising device or inhaler.Alternatively the sealed container may be a unitary dispensing devicesuch as a single dose nasal inhaler or an aerosol dispenser fitted witha metering valve (metered dose inhaler) which is intended for disposalonce the contents of the container have been exhausted.

Where the dosage form comprises an aerosol dispenser, it preferablycontains a suitable propellant under pressure such as compressed air,carbon dioxide or an organic propellant such as a hydrofluorocarbon(HFC). Suitable HFC propellants include 1,1,1,2,3,3,3-heptafluoropropaneand 1,1,1,2-tetrafluoroethane. The aerosol dosage forms can also takethe form of a pump-atomiser. The pressurised aerosol may contain asolution or a suspension of the active compound. This may require theincorporation of additional excipients e.g. co-solvents and/orsurfactants to improve the dispersion characteristics and homogeneity ofsuspension formulations. Solution formulations may also require theaddition of co-solvents such as ethanol.

For pharmaceutical compositions suitable and/or adapted for inhaledadministration, the pharmaceutical composition may be a dry powderinhalable composition. Such a composition can comprise a powder basesuch as lactose, glucose, trehalose, mannitol or starch, the compound ofthe invention or salt thereof (preferably in particle-size-reduced form,e.g. in micronised form), and optionally a performance modifier such asL-leucine or another amino acid and/or metals salts of stearic acid suchas magnesium or calcium stearate. Preferably, the dry powder inhalablecomposition comprises a dry powder blend of lactose e.g. lactosemonohydrate and the compound of the invention or salt thereof. Suchcompositions can be administered to the patient using a suitable devicesuch as the DISKUS® device, marketed by GlaxoSmithKline which is forexample described in GB 2242134 A.

The compounds of the invention thereof may be formulated as a fluidformulation for delivery from a fluid dispenser, for example a fluiddispenser having a dispensing nozzle or dispensing orifice through whicha metered dose of the fluid formulation is dispensed upon theapplication of a user-applied force to a pump mechanism of the fluiddispenser. Such fluid dispensers are generally provided with a reservoirof multiple metered doses of the fluid formulation, the doses beingdispensable upon sequential pump actuations. The dispensing nozzle ororifice may be configured for insertion into the nostrils of the userfor spray dispensing of the fluid formulation into the nasal cavity. Afluid dispenser of the aforementioned type is described and illustratedin WO-A-2005/044354.

A therapeutically effective amount of a compound of the presentinvention will depend upon a number of factors including, for example,the age and weight of the animal, the precise condition requiringtreatment and its severity, the nature of the formulation, and the routeof administration, and will ultimately be at the discretion of theattendant physician or veterinarian. In the pharmaceutical composition,each dosage unit for oral or parenteral administration preferablycontains from 0.01 to 3000 mg, more preferably 0.5 to 1000 mg, of acompound of the invention calculated as the free base. Each dosage unitfor nasal or inhaled administration preferably contains from 0.001 to 50mg, more preferably 0.01 to 5 mg, of a compound of the formula (I) or apharmaceutically acceptable salt thereof, calculated as the free base.

The pharmaceutically acceptable compounds the invention can beadministered in a daily dose (for an adult patient) of, for example, anoral or parenteral dose of 0.01 mg to 3000 mg per day or 0.5 to 1000 mgper day, or a nasal or inhaled dose of 0.001 to 50 mg per day or 0.01 to5 mg per day, of the compound of the formula (I) or a pharmaceuticallyacceptable salt thereof, calculated as the free base. This amount may begiven in a single dose per day or more usually in a number (such as two,three, four, five or six) of sub-doses per day such that the total dailydose is the same. An effective amount of a salt thereof, may bedetermined as a proportion of the effective amount of the compound ofthe invention per se.

The compounds of the invention and may be employed alone or incombination with other therapeutic agents. Combination therapiesaccording to the present invention thus comprise the administration ofat least one compound of the invention or a pharmaceutically acceptablesalt thereof, and the use of at least one other pharmaceutically activeagent. Preferably, combination therapies according to the presentinvention comprise the administration of at least one compound of theinvention or a pharmaceutically acceptable salt thereof, and at leastone other pharmaceutically active agent. The compound(s) of theinvention and the other pharmaceutically active agent(s) may beadministered together in a single pharmaceutical composition orseparately and, when administered separately this may occursimultaneously or sequentially in any order. The amounts of thecompound(s) of the invention and the other pharmaceutically activeagent(s) and the relative timings of administration will be selected inorder to achieve the desired combined therapeutic effect. Thus in afurther aspect, there is provided a combination comprising a compound ofthe invention and at least one other pharmaceutically active agent.

Thus in one aspect, the compound and pharmaceutical compositionsaccording to the invention may be used in combination with or includeone or more other therapeutic agents, for example selected fromantibiotics, anti-virals, glucocorticosteroids, muscarinic antagonistsand beta-2 agonists.

It will be appreciated that when the compound of the present inventionis administered in combination with other therapeutic agents normallyadministered by the inhaled, intravenous, oral or intranasal route, thatthe resultant pharmaceutical composition may be administered by the sameroutes. Alternatively the individual components of the composition maybe administered by different routes.

One embodiment of the invention encompasses combinations comprising oneor two other therapeutic agents.

It will be clear to a person skilled in the art that, where appropriate,the other therapeutic ingredient(s) may be used in the form of salts,for example as alkali metal or amine salts or as acid addition salts, orprodrugs, or as esters, for example lower alkyl esters, or as solvates,for example hydrates, to optimise the activity and/or stability and/orphysical characteristics, such as solubility, of the therapeuticingredient. It will be clear also that, where appropriate, thetherapeutic ingredients may be used in optically pure form.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical composition and thus pharmaceuticalcompositions comprising a combination as defined above together with apharmaceutically acceptable diluent or carrier represent a furtheraspect of the invention.

BACKGROUND EXPERIMENTAL LCMS Methods

Method B

LC/MS (Method B) was conducted on an Acquity UPLC BEH C18 column (50mm×2.1 mm i.d. 1.7 μm packing diameter) at 40 degrees centigrade,eluting with 0.1% v/v solution of Formic Acid in Water (Solvent A) and0.1% v/v solution of Formic Acid in Acetonitrile (Solvent B) using thefollowing elution gradient 0-1.5 min 3-100% B, 1.5-1.9 min 100% B,1.9-2.0 min 3% B at a flow rate of 1 ml/min. The UV detection was asummed signal from wavelength of 210 nm to 350 nm. The mass spectra wererecorded on a Waters ZQ Mass Spectrometer using Alternate-scan Positiveand Negative Electrospray. Ionisation data was rounded to the nearestinteger.

Method D

LC/MS (Method D) was conducted on a Supelcosil LCABZ+PLUS column (3 μm,3.3 cm×4.6 mm ID) eluting with 0.1% HCO₂H and 0.01 M ammonium acetate inwater (solvent A), and 95% acetonitrile and 0.05% HCO₂H in water(solvent B), using the following elution gradient 0-0.7 minutes 0% B,0.7-4.2 minutes 0→100% B, 4.2-5.3 minutes 100% B, 5.3-5.5 minutes 100→0%B at a flow rate of 3 mL/minute. The mass spectra (MS) were recorded ona Fisons VG Platform mass spectrometer using electrospray positiveionisation [(ES+ve to give [M+H]⁺ and [M+NH₄]⁺ molecular ions] orelectrospray negative ionisation [(ES−ve to give [M−H]− molecular ion]modes. Analytical data from this apparatus are given with the followingformat: [M+H]⁺ or [M−H]⁻.

Method E

LC/MS (Method E) was conducted on a Chromolith Performance RP 18 column(100×4.6 mm id) eluting with 0.01M ammonium acetate in water (solvent A)and 100% acetonitrile (solvent B), using the following elution gradient0-4 minutes 0-100% B, 4-5 minutes 100% B at a flow rate of 5 ml/minute.The mass spectra (MS) were recorded on a micromass Platform-LC massspectrometer using atmospheric pressure chemical positive ionisation[AP+ve to give MH+ molecular ions] or atmospheric pressure chemicalnegative ionisation [AP−ve to give (M−H)− molecular ions] modes.Analytical data from this apparatus are given with the following format:[M+H]+ or [M−H]−.

Method F

LC/MS (Method F) was conducted on an Sunfire C18 column (30 mm×4.6 mmi.d. 3.5 μm packing diameter) at 30 degrees centigrade, eluting with0.1% v/v solution of Trifluoroacetic Acid in Water (Solvent A) and 0.1%v/v solution of Trifluoroacetic Acid in Acetonitrile (Solvent B) usingthe following elution gradient 0-0.1 min 3% B, 0.1-4.2 min 3-100% B,4.2-4.8 min 100% B, 4.8-4.9 min 100-3% B, 4.9-5.0 min 3% B at a flowrate of 3 ml/min. The UV detection was an averaged signal fromwavelength of 210 nm to 350 nm and mass spectra were recorded on a massspectrometer using positive electrospray ionization. Ionisation data wasrounded to the nearest integer.

Compound (I) (+)-Phenylmethyl(1-methyl-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl)carbamate

Racemic mixture of phenylmethyl(1-methyl-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl)carbamate [prepared according to the procedure describedin the J. Med. Chem., (1988, 31(1), 176-181)] was separated by HPLCusing a (R,R) whelk-01 column with Hexane/EtOH as the mobile phase. Thesample was prepared in a 80/20 mixture EtOH/Hexane (Note: the samplerequired heating and filtering prior to addition to the column). Thesystem used for preparative separation was as follows: Column: (R,R)Whel-01 51×250 mm column (2 inch columns); mobile phase: 50/50,Hexane/EtOH; Flow rate: 45.0 mL/min; UV wavelength: 254 nm. The titlecompound eluted at 49 min as the first peak. [α]_(D)=+44.7 c=1.0525(g/100 mL)/MeOH. The other enantiomer came off at 58 minutes.

Intermediate 1 [1-(1H-1,2,3-benzotriazol-1-yl)ethyl](4-bromophenyl)amine

To a suspension of benzotriazole (139 g, 1.16 mol) in toluene (2 L) in a3 L, four neck flask under nitrogen atmosphere was added at roomtemperature a solution of 4-bromoaniline (200 g, 1.16 mol) in toluene(300 mL). Then, via a dropping funnel was added drop wise acetaldehyde(64.7 ml, 1.17 mol) in solution in toluene (200 mL). The reactionmixture becomes progressively homogenous and then gives a precipitate.The resulting mixture is stirred 12 hours under nitrogen atmosphere andthen filtered. The precipitate is recrystallised in toluene to affordthe title compound as a white solid (304 g, 82%). 1H NMR (300 MHz,CHLOROFORM-d) δ ppm 2.1 (m, 3H) 4.9 (m, 0.66H) 5.15 (m, 0.33H) 6.5-6.9(m, 3H) 7.2-8.2 (m, 7H)

Intermediate 2(6-bromo-2-methyl-1,2,3,4-tetrahydro-4-quinolinyl)formamide

A 3 L, four neck flask under nitrogen atmosphere was charged withN-vinyl formamide (66.2 g, 0.946 mol) and dry THF (400 mL). BF₃Et₂O (239mL, 1.9 mol) were added dropwise at −5° C. to the milky mixture. After15 minutes Intermediate 1 (150 g, 0.473 mol) in solution in THF (1 L)was added at −5° C. After 2 h, the mixture was slowly and carefullypoured in a NaHCO₃ saturated solution (5 L). Ethyl acetate (2 L) wasadded and the mixture was transferred to a separating funnel. Theorganic layer was separated and was washed 1×200 mL H₂O, 1×200 mL brineand dried (Na₂SO₄). The mixture was filtered and the solids washed 1×50mL ethyl acetate. The filtrate was concentrated progressively until aprecipitate appeared and the mixture cooled in an ice bath during 2 h.The precipitate was filtered through a Buchner funnel, and washed with2×100 mL i-Pr₂O to deliver the title compound as a solid (71 g, 56%).

LC/MS: (Method E), m/z 269 and 271 [M+H]⁺, Rt=2.29 min;

1H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.98 (d, 3H) 1.24 (q, 1H) 2.04(ddd, 1H) 3.33 (m, 1H) 5.17 (m, 1H) 5.45 (m, 1H) 6.15 (d, 1H) 6.88 (dd,1H) 7.00 (d, 1H) 8.11 (s, 1H)

Intermediate 3[1-acetyl-6-bromo-2-methyl-1,2,3,4-tetrahydro-4-quinolinyl]formamide

Acetyl chloride (21 mL, 0.29 mol) is added dropwise at 0° C. to asolution of Intermediate 2 (71 g, 0.26 mol) in a mixture of DCM (1 L)and pyridine (350 mL). After stirring 2 hours at 0° C. the mixture ispoured into a mixture of crushed ice (2 kg) and concentrated HCl (450mL). The product is extracted with DCM (1 L) washed with brine and driedover Na₂SO₄. Concentration under vacuo afforded the expected product asan off white solid (82 g, 100%).

1H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.98 (d, 3H) 1.15 (m, 1H) 1.95 (s,3H) 2.4 (m, 1H) 4.7 (m, 1H) 4.85 (m, 1H) 5.8 (br d, 1H) 6.85 (d, 1H)7.15 (s, 1H) 7.25 (d, 1H) 8.2 (s, 1H)

Intermediate 4 Methyl4-[1-acetyl-4-(formylamino)-2-methyl-1,2,3,4-tetrahydro-6-quinolinyl]benzoate

To a suspension of intermediate 3(1-acetyl-6-bromo-2-methyl-1,2,3,4-tetrahydro-4-quinolinyl)formamide(62.24 g) in DME (600 ml) was added palladium tetrakis (11.56 g) at roomtemperature. After 10 min of stirring, were added{4-[(methyloxy)carbonyl]phenyl}boronic acid (54 g) and a 2N solution ofNa₂CO₃ (300 mL) and the mixture was stirred heated to reflux for 16 h.The mixture was concentrated under reduced pressure. After addition of200 ml of DCM to the residue, the product precipitated, it was filteredand washed with water (3*100 mL). To remove the rest of the water, thesolid was washed with isopropyl ether (100 ml), the solid was then addedto 220 ml of warm isopropyl ether and the resulting mixture was left inthe sonicator. The solid was filtered off and dried to afford the titlecompound as a beige solid (64.7 g)

LCMS (Method E) Rt 2.58 MH+ 367

Intermediate 5 Methyl4-(1-acetyl-4-amino-2-methyl-1,2,3,4-tetrahydro-6-quinolinyl)benzoate

A suspension of Intermediate 4 (20.0 g) in methanol (400 mL) wasrefluxed, then treated with HCl 6N (18 mL). The resulting mixture wasrefluxed for 2 h. The suspension was filtered off on whatman and thefiltrate was concentrated until dryness. Acetone (70 mL) was added tothe residue, the solid was filtered off and dried. The resulting salt inethyl acetate (300 mL) was treated with NaOH 1N (100 ml). Aqueous andorganic layers were separated. Aqueous layer was extracted withCH₂Cl₂/MeOH 9:1 (300 mL). The organic layers were combined, dried andconcentrated until dryness to give the title compound as a white solid(13.83 g). LCMS (Method E) Rt 2.51 MH+ 339

Intermediate 6 (2S,3S)-2,3-bis[(phenylcarbonyl)oxy]butanedioicacid-methyl4-(1-acetyl-4-amino-2-methyl-1,2,3,4-tetrahydro-6-quinolinyl)benzoate(1:2)

A mixture of the racemic amine Intermediate 5 (185 g,) in EtOH (600 mL)and L-(+)-lactic acid (20% in water, 450 mL) was heated to reflux during30 minutes. After concentration under reduced pressure hexane (300 mL)was added to the residue and the resulting mixture heated to reflux 10min. The mixture was allowed to settle and the hexane phase wasdiscarded. The remaining paste was taken up with Et₂O (300 mL), heatedto reflux during 10 minutes and allowed to settle. The Et₂O phase wasdiscarded and the resulting paste once again was treated with hexane(200 mL), heated to reflux and allowed to settle.

The hexane phase was discarded and EtOAc (2.3 L) was added to theremaining paste. The mixture was heated to reflux and allowed to standat room temperature for 16 hours. The precipitate was filtered andwashed with EtOAc (200 mL). The filtrate was made basic with addition ofNa₂CO₃ and the resulting free amino was extracted with EtOAc (3×1000mL), washed with water, dried over Na₂SO₄ and concentrated under reducedpressure. The resulting free amino (95 g) in solution in THF (950 mL)was treated with L(−)-dibenzoyltartaric acid (50.3 g, 0.14 mol) andheated to reflux 30 minutes. The resulting precipitate was allowed tostand at room temperature during 16 hours and then was filtered andwashed with THF (200 ml). An HPLC monitoring of a neutralised aliquotindicated a 95.6% ee of the expected amine enantiomer. Recrystallisationof the tartaric salt in EtOH (1 L) afforded the title compound (95 g) asa single diastereomer salt. mp:196° C.

1H NMR (300 MHz, DMSO-d6) δ ppm 0.95 (d, 3H) 1.15 (m, 1H) 2.05 (s, 3H)2.55 (m, 1H) 3.85 (s, 3H) 4.0 (m, 1H) 4.55 (m, 1H) 5.7 (s, 1H, CHtartaric) 7.4 (m, 3H) 7.6 (m, 2H) 7.85 (m, 3H), 7.95 (m, 4H).

Intermediate 7 Methyl4-[(2S,4R)-1-acetyl-4-amino-2-methyl-1,2,3,4-tetrahydro-6-quinolinyl]benzoate

A mixture of Intermediate 6 (121 g) in DCM (3 L) was made basic withaddition of an aqueous solution of Na₂CO₃. The resulting free amine wasextracted with DCM (2 L) washed with water and dried over Na₂SO₄ todeliver the title compound as an off white solid (79 g).

1H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.15 (m, 4H) 1.7 (m, 2H) 2.15 (s,3H) 2.6 (m, 1H) 3.8 (dd, 1H), 3.95 (s, 3H) 4.85 (m, 1H) 7.2 (d, 1H) 7.55(d, 1H) 7.7 (d, 2H), 7.8 (s, 1H) 8.1 (d, 2H)

[α]_(D)=+333.8 (c=0.985 g/cl, EtOH).

The title compound eluted at 18.57 min by HPLC as the second peak usinga CHIRACEL OD (250×4.6 mm 10 μm) column with hexane/ethanol 80/20 as themobile phase. A 1 ml/mn flow rate was applied and 10 μL of sampleprepared with the dilution of 1 mg of the title compound in 1 ml ofeluent was injected. Detection of the compound was carried out with both210 and 254 nM UV wavelengths. The other enantiomer came off at 12.8min.

Intermediate 8 Methyl4-{(2S,4R)-1-acetyl-4-[(4-chlorophenyl)amino]-2-methyl-1,2,3,4-tetrahydro-6-quinolinyl}benzoate

To a flask charged with the Intermediate 7 (800 mg, 2.4 mmol) in toluene(20 mL) was added 4-chlorobromobenzene (501 mg, 2.6 mmol), Pd₂(dba)₃ (87mg, 0.09 mmol), NaO^(t)Bu (319 mg, 3.3 mmol) and2′-(dicyclohexylphosphanyl)-N,N-dimethyl-2-biphenylamine (74 mg, 0.19mmol). The resulting mixture was stirred to 80° C. during 16 hours and 3additional hours at reflux. The mixture was poured into water and wasmade acidic upon addition of 1N HCl. Extraction was carried out withEtOAc (2×75 ml) and the organic layers were washed with water and driedover Na₂SO4. After filtration, concentration under reduced pressure andpurification by column chromatography eluting with C₆H₁₂/EtOAc:80/20 thetitle compound was obtained as a white solid (350 mg).

1H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.2 (d, 3H) 1.35 (m, 1H) 2.25 (s,3H) 2.7 (m, 1H) 3.95 (s, 3H), 4.25 (m, 1H) 4.95 (m, 1H) 6.6 (d, 2H) 7.15(d, 2H) 7.25 (s, 1H) 7.55 (m, 4H), 8.1 (d, 2H)

LC/MS (Method D): m/z 449 [M+H]⁺ and 447 [M−H]⁻ Rt=3.67 min.[α]_(D)=+326 (c=0.98 g/cl, EtOH)

The title compound eluted at 22.58 min by HPLC as the second peak usinga CHIRACEL OD (250×4.6 mm 10 μm) column with hexane/ethanol 90/10 as themobile phase. A 1 ml/mn flow rate was applied and 10 μL of sampleprepared with the dilution of 1 mg of the title compound in 1 ml ofeluent was injected. Detection of the compound was carried out with both210 and 254 nM UV wavelengths. The other enantiomer came off at 15.46min.

Compound (II)4-{(2S,4R)-1-acetyl-4-[(4-chlorophenyl)amino]-2-methyl-1,2,3,4-tetrahydro-6-quinolinyl}benzoicacid

A solution of Intermediate 8 (320 mg, 0.73 mmol) in EtOH (10 ml) and 1NNaOH (1.5 ml, 1.5 mmol) was heated to reflux. After 1 hour a tlcmonitoring indicated the completion of the reaction. The crude mixturewas evaporated to dryness and the residue taken up in water (10 mL).Acidification of the mixture at pH=3 was carried out by addition of a 1NHCl solution. The organic materials were extracted with EtOAc (3×25 mL)and the organic phase combined and washed with brine and dried overNa₂SO₄. After concentration under vacuo the residue was taken up in aDCM/hexane mixture to give a red solid after filtration. The compoundwas recrystallised in EtOAC, filtered and washed with i-Pr₂O. Theresulting white powder was solubilised in MeOH/H₂O, concentrated todryness and taken up with H₂O. Finally filtration of the precipitateafforded the title compound as a white powder (147 mg), mp: 275° C.

HRMS calculated for C₂₅H₂₃N₂O₃Cl (M−H)⁻ 433.1319, found: 433.1299. Rt:2.21 min. 1H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.2 (d, 3H) 1.35 (m, 1H)2.3 (s, 3H) 2.7 (m, 1H) 4.25 (dd, 1H) 4.95 (m, 1H) 6.65 (d, 2H) 7.15 (d,2H) 7.25 (s, 1H) 7.55 (m, 4H), 8.15 (d, 2H)

[α]_(D)=+395 (c=0.96 g/cl, EtOH) measured at the EtOAc recrystallisationstage.

The title compound eluted at 4.51 min by HPLC as the first peak using aChiralpak IA (250×4.6 mm 5 μm) column with tert-butyl methyl oxide(MTBE) +0.1% TFA/Ethanol:90/10 as the mobile phase. A 1 ml/mn flow ratewas applied and 10 L of sample prepared with the dilution of 1 mg of thetitle compound in 1 ml of eluent was injected. Detection of the compoundwas carried out with both 210 and 254 nM UV wavelengths. The otherenantiomer came off at 5.92 min.

Compound (III) 1-Acetyl-2-methyl-1,2,3,4-tetrahydroquinoline

2-Methyl-1,2,3,4-tetrahydroquinoline (216 mg, 1.469 mmol, available fromTCl), was measured into a reaction test tube and acetic anhydride (0.139ml, 1.469 mmol) was added and left to stir overnight. LCMS analysisshowed the reaction had gone to completion. The crude product waspurified by MDAP to give the desired compound (677 mg)

LCMS (Method B). RT 0.93, MH+ 190

Intermediate 9 1-methylethyl (2E)-2-butenoylcarbamate

Isopropyl carbamate (30 g, 291 mmol, available from TCl) was charged toa 3 L Lara vessel and dry Tetrahydrofuran (THF) (150 ml) added.(2E)-2-butenoyl chloride (31.2 ml, 326 mmol, available from Aldrich) wasadded under Nitrogen and the jacket cooled to −30° C. When the solutiontemperature reached −17° C. 1M Lithium tert-butoxide (655 ml, 655 mmol)was added by peristaltic pump over 2 hours, keeping the reactiontemperature between −10° C. and −18° C. Once the addition was completethe mixture was complete the mixture was stirred for 30 mins and broughtto 0° C. Diethyl ether (450 ml) and 1M HCl (375 ml) were added and themixture brought to 20° C. with vigourous stirring. The stirring wasstopped, the layers allowed to separate and the aqueous layer run off.Brine (375 ml) was added and the mixture stirred vigourously. Thestirring was stopped, the layers allowed to separate and the aqueouslayer run off. The organic layer was dried (magnesium sulfate), filteredand evaporated to a brown oil (60 g). The mixture was loaded on to a40+M Biotage silica column and eluted with DCM:ethyl acetate (1:1 to0:1, 10 CV). The product containing fractions were evaporated to drynessand loaded on to a 1500 g Redisep Isco silica column and eluted with agradient of 0 to 40% ethyl acetate in cyclohexane. The clean, productcontaining fractions were evaporated to an off white solid (15.41 g).LCMS (Method C): Rt=0.68, MH+=172

Intermediate 10 (R-BINAP)ditriflatebis(acetonitrile)palladium(II)

R-(+)-BINAP (6.08 g, 9.76 mmol, available from Avocado) was stirred inDichloromethane (DCM) (626 ml) and dichlorobis(acetonitrile)palladium(II) (2.5 g, 9.64 mmol, available from Aldrich) added. The mixture wasstirred under Nitrogen for 30 mins, the suspension had not become asolution and more DCM (100 ml) was added. The mixture was stirred for afurther 30 mins and Silver triflate (5.00 g, 19.47 mmol, available fromAldrich) dissolved in Acetonitrile (250 ml) was added. The mixturechanged from an orange cloudy suspension to a yellow suspension. Themixture was stirred for 1 hour, filtered through celite and evaporatedto an orange solid. The residue was dried under vacuum (at approximately14 mbar) at room temperature over the weekend to give the desiredproduct (10.69 g).

1H NMR (400 MHz, MeCN-d3) δ ppm 2.0 (s, 6H), 6.7 (d, 2H), 6.9 (br m,4H), 7.1 (br t, 2H), 7.2 (t, 2H), 7.5-7.9 (m, 22H)

Intermediate 11 (3S)-3-(phenylamino)butanenitrile

(3S)-3-aminobutanenitrile (8.6 g, 102 mmol, may be prepared as describedin PCT Int. Appl., 2005100321), bromobenzene (16.16 ml, 153 mmol) andcesium carbonate (50.0 g, 153 mmol) were combined in Toluene (100 ml)under nitrogen were stirred for 45 mins. Phenylboronic acid (0.187 g,1.534 mmol, Aldrich), palladium(II) acetate (0.188 g, 0.837 mmol,available from Aldrich) and2-dicyclohexylphosphino-2′-(N,N-dimethylamino)biphenyl (0.443 g, 1.125mmol, available from Aldrich) were combined in Tetrahydrofuran (THF)(6.67 ml) under nitrogen and stirred for 45 mins. The THF solution wasadded to the toluene solution and the reaction heated to 80° C.overnight. The reaction mixture was cooled and partitioned between EtOAc(500 ml) and water (300 ml). The aqueous layer was reextracted withEtOAc (200 ml). The combined organic layers were washed with water andbrine (500 ml each) and then dried with Na2SO4, filtered andconcentrated to yield an orange oil. The crude product was taken up inthe minimum of DCM, applied to a 330 g Companion XL column and elutedwith 5% Ethyl Acetate in cyclohexane for 1 CV then 5-30% Ethyl Acetateover 12 CV then held at 30% for 3 CV; UV collection; 450 ml fractions.The product was isolated as an off-white solid (11.3526 g).

LCMS (Method B): Rt=0.87, MH+=161

Intermediate 12 (3S)-3[(4-bromophenyflamino]butanenitrile

(3S)-3-(phenylamino)butanenitrile (for a preparation see Intermediate11) (11.3526 g, 70.9 mmol) was taken up in N,N-Dimethylformamide (DMF)(200 mL) under nitrogen and cooled in an ice-bath. NBS (12.61 g, 70.9mmol) was added and the reaction stirred. After 20 mins, the reactionwas partitioned between EtOAc (1000 ml) and water (500 ml). The organiclayer was washed with 2M NaOH×2, water and brine (500 ml each) and thendried with Na2SO4, filtered and concentrated to yield the product as acream solid (17.3 g). LCMS (Method B): Rt=1.05, MH+=239

Intermediate 13 (3S)-3-[(4-bromophenyl)amino]butanamide

(3S)-3-[(4-bromophenyl)amino]butanenitrile (for a preparation seeIntermediate 12) (17.3 g, 72.4 mmol) was taken up in Toluene (500 ml)and H2SO4 (19.28 ml, 362 mmol) added. The biphasic mixture was stirredat 60° C. After two hours, only a small amount of SM remained by LCMS sothe reaction was diluted with water (500 ml) and the phases separated.The aqueous phase was basified with 10N NaOH and extracted with EtOAc(2×750 ml). The combined organics were dried with Na2SO4, filtered andconcentrated to yield the product as a cream solid (17.5 g). LCMS(Method B): Rt=0.77, MH+=257

Intermediate 14 1-methylethyl{(3S)-3-[(4-bromophenyl)amino]butanoyl}carbamate

(3S)-3-[(4-bromophenyl)amino]butanamide (for a preparation seeIntermediate 13, 24.9 g, 97 mmol) was taken up in Ethyl acetate (850 mL)and cooled to <−9° C. (internal). Isopropyl chloroformate (116 mL, 116mmol, Aldrich) was added followed by slow addition of Lithiumtert-butoxide (18.61 g, 232 mmol) in Tetrahydrofuran (THF) (232 mL)keeping the temperature below 0° C. The reaction was stirred for 30 minsthen checked by LCMS which showed a complete reaction. The mixture waspartitioned between EtOAc (1000 ml) and 2N HCl (2000 ml). The organiclayer was washed with brine (2000 ml) and then dried with Na2SO4,filtered and concentrated to yield the product as a brown oil (17.9 g)LCMS (Method B): Rt=1.09, MH+=343

Alternative Method

1-methylethyl (2E)-2-butenoylcarbamate (Intermediate 9, 9.38 g, 54.8mmol) was stirred in Toluene (281 ml) under nitrogen and(R-BINAP)ditriflatebis(acetonitrile)palladium(II) (Intermediate 10, 3.35g, 3.01 mmol) added. The catalyst formed a gummy ball, the solutionturned to an opaque yellow mixture and was stirred for 20 mins.4-bromoaniline (14.14 g, 82 mmol) was added, the solution turned a clearlight brown and the gummy catalyst dissolved further. The mixture wasstirred overnight,

Similarly a second batch of 1-methylethyl (2E)-2-butenoylcarbamate(Intermediate 9, 8.51 g, 49.7 mmol) was stirred in Toluene (255 ml)under nitrogen and (R-BINAP)ditriflatebis(acetonitrile)palladium(II)(3.04 g, 2.73 mmol) added. The catalyst formed a gummy ball, thesolution turned to an opaque yellow mixture and was stirred for 20 mins.4-bromoaniline (12.83 g, 74.6 mmol) was added, the solution turned aclear light brown and the gummy catalyst dissolved further. The mixturewas stirred overnight. The two reaction mixtures were combined andloaded on to a 1.5 kg Isco silica Redisep column. The column was elutedwith DCM:MeOH (0%->0.5%, 19 CV). The clean, product containing fractionswere evaporated to a pale brown oil. The mixture was dried in a vacuumoven overnight at 40° C. to give a white solid (24.2 g, 67% overall).

LCMS (Method C): Rt=0.91, MH+=343. ee=92%.

Intermediate 15 1-methylethyl[(2S,4R)-6-bromo-2-methyl-1,2,3,4-tetrahydro-4-quinolinyl]carbamate

1-methylethyl {(3S)-3-[(4-bromophenyl)amino]butanoyl}carbamate (for apreparation see Intermediate 14) (17.9 g, 52.2 mmol) was taken up inEthanol (150 mL) and cooled to below −10° C. (internal) in a CO2/acetonebath. NaBH4 (1.381 g, 36.5 mmol) was added followed by MagnesiumChloride hexahydrate (11.35 g, 55.8 mmol) in Water (25 mL) keeping thetemperature below −5° C. The mixture was allowed to stir at <0° C. for 1hr then warmed to RT and stirred for an hour. The resulting thicksuspension was poured into a mixture of citric acid (25.05 g, 130 mmol),HCl (205 mL, 205 mmol) and Dichloromethane (DCM) (205 mL). The biphasicmixture was stirred at RT for 1 hr. LCMS showed no SM remained so theorganic layer was extracted and dried with Na2SO4, filtered andconcentrated to yield the product as a light brown solid (14.1 g). LCMS(Method B): Rt=1.13, MH+=327

Intermediate 16 1-methylethyl[(2S,4R)-1-acetyl-6-bromo-2-methyl-1,2,3,4-tetrahydro-4-quinolinyl]carbamate

1-methylethyl[(2S,4R)-6-bromo-2-methyl-1,2,3,4-tetrahydro-4-quinolinyl]carbamate (fora preparation see Intermediate 15) (14.1 g, 43.1 mmol) was taken up inDichloromethane (DCM) (400 mL) under nitrogen at RT. Pyridine (10.46 mL,129 mmol) then Acetyl chloride (4.60 mL, 64.6 mmol) were added and thereaction stirred overnight. LCMS showed a complete reaction so it waspartitioned between EtOAc (2000 ml) and sat. NaHCO₃ (800 ml). Theorganic layer was extracted and washed with water and brine (1500 mleach) and then dried with Na₂SO₄, filtered and concentrated to yield apurple solid. The crude product was taken up in the minimum of DCM andapplied to a 330 g Companion XL column and eluted with a gradient of12-63% Ethyl Acetate in cyclohexane. Product containing fractions werecollected as an off-white solid (12.37 g).

LCMS (Method B): Rt=1.03, MH+=369

[alpha]D=+281.1025° (T=20.7° C., 10 mm cell, c=0.508 g/100 ml, ethanol).

Intermediate 17 1-methylethyl[(2S,4R)-1-acetyl-6-(4-formylphenyl)-2-methyl-1,2,3,4-tetrahydro-4-quinolinyl]carbamate

1-methylethyl[(2S,4R)-1-acetyl-6-bromo-2-methyl-1,2,3,4-tetrahydro-4-quinolinyl]carbamate(for a preparation see Intermediate 16) (1 g, 2.71 mmol),(4-formylphenyl)boronic acid (0.487 g, 3.25 mmol, available fromAldrich), Pd(Ph₃P)₄, (0.156 g, 0.135 mmol) and potassium carbonate(0.487 g, 3.52 mmol) were combined in dry ethanol (7 ml) and dry toluene(7.00 ml) and the reaction mixture was de-gassed for 10 mins. Thereaction mixture was heated at 85° C. overnight. The reaction mixturewas allowed to cool to r.t. and concentrated. The crude reaction mixturewas partitioned between water (15 ml) and ethyl acetate (5 ml) andstirred at r.t. for 30 mins. A light grey solid precipitated out and wasfiltered off, washed with water (5 ml) and dried in a vacuum oven togive 854 mg of grey solid. LCMS (Method B): Rt=1.00, MH+=395

Compound (IV) 1-methylethyl((2S,4R)-1-acetyl-2-methyl-6-{4-[methylamino)methyl]phenyl}-1,2,3,4-tetrahydro-4-quinolinyl)carbamate

1-methylethyl[(2S,4R)-1-acetyl-6-(4-formylphenyl)-2-methyl-1,2,3,4-tetrahydro-4-quinolinyl]carbamate(for a preparation see Intermediate 17) (100 mg, 0.254 mmol) wasdissolved in Methanol (3 mL) and 2M methylamine in THF (0.254 mL, 0.507mmol) was added. The yellow solution was stirred under nitrogen at roomtemperature for 135 minutes at which point sodium borohydride (15.35 mg,0.406 mmol) was added. The reaction was stirred for 1 h then leftsitting overnight. The reaction was quenched with sat. aqueous sodiumbicarbonate solution (1 mL) and EtOAc (8 mL) was added. A white solidwas filtered off (bond elut reservoir) and found to be the desiredproduct (34 mg). The filtrate was partitioned and the organic layerdried. Concentration of the organic layer gave 67 mg of a colourlessresidue which was applied to a silica 12+S Biotage column and purifiedeluting with a gradient of 1-5% methanolic ammonia in DCM. Concentrationof the product containing fractions gave another batch of the desiredproduct (52 mg).

LCMS (Method C): Rt 0.71, MH+=410

1H NMR (CHLOROFORM-d, 600 MHz): δ (ppm) 7.55 (d, J=8.1 Hz, 2H), 7.49 (d,J=7.9 Hz, 1H), 7.45 (br. s., 1H), 7.41 (d, J=7.9 Hz, 2H), 7.18 (d, J=7.3Hz, 1H), 4.52-5.08 (m, 4H), 3.81 (s, 2H), 2.62 (ddd, J=12.5, 8.3, 4.5Hz, 1H), 2.50 (s, 3H), 2.17 (s, 3H), 1.21-1.37 (m, 7H), 1.18 (d, J=6.4Hz, 3H)

Reference Compound A 2-methyl-6-(methyloxy)-4H-3,1-benzoxazin-4-one

A solution of 5-methoxyanthranilic acid (Lancaster) (41.8 g, 0.25 mol)was refluxed in acetic anhydride (230 mL) for 3.5 h before beingconcentrated under reduced pressure. The crude compound was thenconcentrated twice in the presence of toluene before being filtered andwashed twice with ether to yield to the title compound (33.7 g, 71%yield) as a brown solid; LC/MS (Method D): m/z 192 [M+H]⁺, Rt 1.69 min.

Reference Compound B[2-amino-5-(methyloxy)phenyl](4-chlorophenyl)methanone

To a solution of 2-methyl-6-(methyloxy)-4H-3,1-benzoxazin-4-one (for apreparation see Reference compound A) (40.0 g, 0.21 mol) in atoluene/ether (2/1) mixture (760 mL) at 0° C. was added dropwise asolution of 4-chlorophenylmagnesium bromide (170 mL, 1M in Et₂O, 0.17mol). The reaction mixture was allowed to warm to room temperature andstirred for 1 h before being quenched with 1N HCl (200 mL). The aqueouslayer was extracted with EtOAc (3×150 mL) and the combined organics werewashed with brine (100 mL), dried over Na₂SO₄, filtered and concentratedunder reduced pressure. The crude compound was then dissolved in EtOH(400 mL) and 6N HCl (160 mL) was added. The reaction mixture wasrefluxed for 2 h before being concentrated to one-third in volume. Theresulting solid was filtered and washed twice with ether before beingsuspended in EtOAc and neutralised with 1N NaOH. The aqueous layer wasextracted with EtOAc (3×150 mL) and the combined organics were washedwith brine (150 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure. The title compound was obtained as a yellow solid (39g, 88% yield); LC/MS (Method D): m/z 262 [M+H]⁺, Rt 2.57 min.

Reference Compound C MethylN¹-[2-[(4-chlorophenyl)carbonyl]-4-(methyloxy)phenyl]-N²-{[(9H-fluoren-9-ylmethyl)oxy]carbonyl}-L-α-asparaginate

Methyl N-{[(9H-fluoren-9-ylmethyl)oxy]carbonyl}-L-α-aspartyl chloride(Int. J. Peptide Protein Res. 1992, 40, 13-18) (93 g, 0.24 mol) wasdissolved in CHCl₃ (270 mL) and[2-amino-5-(methyloxy)phenyl](4-chlorophenyl)methanone (53 g, 0.2 mol)(for a preparation see Reference compound B) was added. The resultingmixture was stirred at 60° C. for 1 h before being cooled andconcentrated at 60% in volume. Ether was added at 0° C. and theresulting precipitate was filtered and discarded. The filtrate wasconcentrated under reduced pressure and used without furtherpurification.

Reference Compound D Methyl[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-oxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetate

To a solution of methylN1-[2-[(4-chlorophenyl)carbonyl]-4-(methyloxy)phenyl]-N2-{[(9H-fluoren-9-ylmethyl)oxy]carbonyl}-L-α-asparaginate(for a preparation see Reference compound C) (assumed 0.2 mol) in DCM(500 mL) was added Et₃N (500 mL, 3.65 mol) and the resulting mixture wasrefluxed for 24 h before being concentrated. The resulting crude aminewas dissolved in 1,2-DCE (1.5 L) and AcOH (104 mL, 1.8 mol) was addedcarefully. The reaction mixture was then stirred at 60° C. for 2 hbefore being concentrated in vacuo and dissolved in DCM. The organiclayer was washed with 1N HCl and the aqueous layer was extracted withDCM (×3). The combined organic layers were washed twice with water, andbrine, dried over Na₂SO₄, filtered and concentrated under reducedpressure. The crude solid was recrystallised in MeCN leading to thetitle compound (51 g) as a pale yellow solid. The filtrate could beconcentrated and recrystallised in MeCN to give to another 10 g of thedesired product R_(f)=0.34 (DCM/MeOH: 95/5).

HRMS (M+H)⁺ calculated for C₁₉H₁₈ ³⁵ClN₂O₄ 373.0955; found 373.0957.

Reference Compound E Methyl[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-thioxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetate

A suspension of P₄S₁₀ (36.1 g, 81.1 mmol) and Na₂CO₃ (8.6 g, 81.1 mmol)in 1,2-DCE (700 mL) at room temperature was stirred for 2 h beforemethyl[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-oxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetate(for a preparation see Reference compound D) (16.8 g, 45.1 mmol) wasadded. The resulting mixture was stirred at 70° C. for 2 h before beingcooled and filtered. The solid was washed twice with DCM and thefiltrate washed with sat. NaHCO₃ and brine. The organic layer was driedover Na₂SO₄, filtered and concentrated under reduced pressure. The crudeproduct was purified by flash-chromatography on silica gel (DCM/MeOH:99/1) to afford the title compound (17.2 g, 98% yield) as a yellowishsolid. LC/MS (Method D): m/z 389 [M(³⁵Cl)+H]⁺, Rt 2.64 min

HRMS (M+H)⁺ calculated for C₁₉H₁₈ ³⁵ClN₂O₃S 389.0727. found 389.0714.

Reference compound F Methyl[(3S)-2-[(1Z)-2-acetylhydrazino]-5-(4-chlorophenyl)-7-(methyloxy)-3H-1,4-benzodiazepin-3-yl]acetate

To a suspension of methyl[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-thioxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetate(for a preparation see Reference compound E (9.0 g, 23.2 mmol) in THF(300 mL) at 0° C. was added hydrazine monohydrate (3.4 mL, 69.6 mmol)dropwise. The reaction mixture was stirred for 5 h between 5° C. and 15°C. before being cooled at 0° C. Et₃N (9.7 mL, 69.6 mmol) was then addedslowly and acetyl chloride (7.95 mL, 69.6 mmol) was added dropwise. Themixture was then allowed to warm to room temperature for 16 h beforebeing concentrated under reduced pressure. The crude product wasdissolved in DCM and washed with water. The organic layer was dried overNa₂SO₄, filtered and concentrated in vacuo to give the crude titlecompound (9.7 g, 98% yield) which was used without further purification.R_(f)=0.49 (DCM/MeOH: 90/10).

Reference Compound G Methyl[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetate

The crude methyl[(3S)-2-[(1Z)-2-acetylhydrazino]-5-(4-chlorophenyl)-7-(methyloxy)-3H-1,4-benzodiazepin-3-yl]acetate(for a preparation see Reference compound F) (assumed 9.7 g) wassuspended in THF (100 ml) and AcOH (60 mL) was added at roomtemperature. The reaction mixture was stirred at this temperature for 2days before being concentrated under reduced pressure. The crude solidwas triturated in i-Pr₂O and filtered to give the title compound (8.7 g,91% over 3 steps) as an off-white solid.

HRMS (M+H)⁺ calculated for C₂₁H₂₀ClN₄O₃ 411.1229; found 411.1245.

Reference Compound H[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]aceticacid

To a solution of Methyl[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetate(for a preparation see Reference compound G) (7.4 g, 18.1 mmol) in THF(130 mL) at room temperature was added 1N NaOH (36.2 mL, 36.2 mmol). Thereaction mixture was stirred at this temperature for 5 h before beingquenched with 1N HCl (36.2 mL) and concentrated in vacuo. Water is thenadded and the aqueous layer was extracted with DCM (×3) and the combinedorganic layers were dried over Na₂SO₄, filtered and concentrated underreduced pressure to give the title compound (7 g, 98% yield) as a paleyellow solid.

Reference Compound I 1,1-dimethylethyl[5-({[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetyl}amino)pentyl]carbamate

A mixture of[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]aceticacid (for a preparation see Reference compound H) (1.0 g, 2.5mmol), HATU(1.9 g, 5 mmol) and DIPEA (0.88 ml, 5 mmol) was stirred for 80 minutesat room temperature, to this was added 1,1-dimethylethyl(4-aminobutyl)carbamate (1.05 ml, 5.0 mmol, available from Aldrich). Thereaction mixture was stirred at room temperature for 2 h before it wasconcentrated. The residue was taken up in dichloromethane and washedwith 1N HCl. The aqueous layer was extracted with dichloromethane twice.Organic layer was washed with 1N sodium hydroxide, followed by asaturated solution of sodium chloride, dried over sodium sulphate andconcentrated. The residue was purified by flash-chromatography on silicausing dichloromethane/methanol 95/5 to give the title compound as ayellow solid (1.2 g). LC/MS (Method D): rt=3.04 min.

Reference Compound JN-(5-aminopentyl)-2-[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetamidetrifluoroacetate

To a solution of 1,1-dimethylethyl[5-({[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetyl}amino)pentyl]carbamate(for a preparation see Reference compound I) (0.2 g, 0.34 mmol) indichloromethane (3 ml) was added trifluoroacetic acid (0.053 ml, 0.68mmol) dropwise at 0° C. The reaction mixture was stirred for 3 h from 0°C. to room temperature. The reaction mixture was concentrated to drynessto afford the title compound as a hygroscopic yellow oil (200 mg)

LC/MS (Method D): rt=2.33 min.

HRMS (M+H)⁺ calculated for C₂₅H₂₉ClN₆O₂ 481.2119; found 481.2162.

Reference Compound K Mixture of 5- and 6-isomers of Alexa Fluor488-N-(5-aminopentyl)-2-[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetamide

N-(5-aminopentyl)-2-[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetamidetrifluoroacetate (for a preparation see Reference compound J) (7.65 mg,0.013 mmol) was dissolved in N,N-Dimethylformamide (DMF) (300 μl) andadded to Alexa Fluor 488 carboxylic acid succinimidyl ester (5 mg, 7.77μmol, mixture of 5 and 6 isomers, available from Invitrogen, productnumber A-20100) in an Eppendorf centrifuge tube. Hunig's base (7.0 μl,0.040 mmol) was added and the mixture vortex mixed overnight. After 18 hthe reaction mixture was evaporated to dryness and the residueredissolved in DMSO/water (50%, <1 ml total), applied to a preparativePhenomenex Jupiter C18 column and eluted with a gradient of 95% A: 5% Bto 100% B (A=0.1% trifluoroacetic acid in water, B=0.1% TFA/90%acetonitrile/10% water) at a flow rate of 10 ml/min over 150 minutes.Impure fractions were combined and re-purified using the same system.Fractions were combined and evaporated to yield the title product (2.8mg) as a mixture of the 2 regioisomers shown. LC/MS (Method F): MH+=999,rt=1.88 min.

Biological Test Methods Fluorescence Anisotropy Binding Assay

The binding of Compounds (I) to (IV) to Bromodomains BRD-2, BRD-3 andBRD-4 was assessed using a Fluorescence Anisotropy Binding Assay.

The Bromodomain protein, fluorescent ligand (Reference compound K seeabove) and a variable concentration of test compound are incubatedtogether to reach thermodynamic equilibrium under conditions such thatin the absence of test compound the fluorescent ligand is significantly(>50%) bound and in the presence of a sufficient concentration of apotent inhibitor the anisotropy of the unbound fluorescent ligand ismeasurably different from the bound value.

All data was normalized to the mean of 16 high and 16 low control wellson each plate. A four parameter curve fit of the following form was thenapplied:y=a+((b−a)/(1+(10^x/10^c)^d)Where ‘a’ is the minimum, ‘b’ is the Hill slope, ‘c’ is the pIC₅₀ and‘d’ is the maximum.

Recombinant Human Bromodomains (BRD-2 (1-473), BRD-3 (1-435) and BRD-4(1-477)) were expressed in E. coli cells (in pET15b vector) with asix-His tag at the N-terminal. The His-tagged Bromodomain was extractedfrom E. coli cells using 0.1 mg/ml lysozyme and sonication. TheBromodomain was then purified by affinity chromatography on a HisTRAP HPcolumn, eluting with a linear 10-500 mM Imidazole gradient, over 20 Cv.Further purification was completed by Superdex 200 prep grade sizeexclusion column. Purified protein was stored at −80 C in 20 mM HEPES pH7.5 and 100 mM NaCl.

Protocol for Bromodomain BRD-2:

All components were dissolved in buffer composition of 50 mM HEPESpH7.4, 150 mm NaCl and 0.5 mM CHAPS with final concentrations of BRD-2,75 nM, fluorescent ligand 5 nM. 10 μl of this reaction mixture was addedusing a micro multidrop to wells containing 100 nl of variousconcentrations of test compound or DMSO vehicle (1% final) in Greiner384 well Black low volume microtitre plate and equilibrated in dark 60mins at room temperature. Fluorescence anisotropy was read in Envision(λex=485 nm, λEM=530 nm; Dichroic −505 nM).

Protocol for Bromodomain BRD-3:

All components were dissolved in buffer of composition 50 mM HEPES pH7.4, 150 mm NaCl and 0.5 mM CHAPS with final concentrations of BRD-3 75nM, fluorescent ligand 5 nM. 10 μl of this reaction mixture was addedusing a micro multidrop to wells containing 100 nl of variousconcentrations of test compound or DMSO vehicle (1% final) in Greiner384 well Black low volume microtitre plate and equilibrated in dark 60mins at room temperature. Fluorescence anisotropy was read in Envision(λex=485 nm, λEM=530 nm; Dichroic −505 nM).

Protocol for Bromodomain BRD-4:

All components were dissolved in buffer of composition 50 mM HEPES pH7.4, 150 mm NaCl and 0.5 mM CHAPS with final concentrations of BRD-4 75nM, fluorescent ligand 5 nM. 10 μl of this reaction mixture was addedusing a micro multidrop to wells containing 100 nl of variousconcentrations of test compound or DMSO vehicle (1% final) in Greiner384 well Black low volume microtitre plate and equilibrated in dark 60mins at room temperature. Fluorescence anisotropy was read in Envision(λex=485 nm, λEM=530 nm; Dichroic −505 nM).

Compounds (I), (II) and (IV) had a pIC₅₀≧6.0 in each of the BRD-2, BRD-3and BRD-4 assays described above.

Compound (III) had a pIC₅₀≦4.3 in each of the BRD-2, BRD-3 and BRD-4assays described above.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The invention claimed is:
 1. A process for the identification of acompound which inhibits the binding of the second bromodomain of each ofhuman BRD-2, BRD-3, and BRD-4 to acetylated lysine residues of theirphysiological partner proteins which process comprises the steps of: 1)selecting compounds with a molecular weight in the range 100 to 750 Da;and 2) determining which of those selected compounds is an interactingcompound, wherein an interacting compound will: a) form a hydrogenbonding interaction in which the compound accepts a hydrogen bond fromthe sidechain NH₂ group of the asparagine residue found at: ASN429 ofBRD-2 BD2, or ASN391 of BRD-3 BD2, or ASN433 of BRD-4 BD2 and b) form aVan der Waals interaction with a lipophilic binding region of a bindingpocket such that one or more heavy atoms of the said compounds liewithin a 7.5 Å range of at least one heavy atom of each of the 3residues of the second bromodomain wherein said bromodomain residues areselected from the group consisting of: PRO371, ASP434, and VAL435 ofBRD-2 BD2; PRO333, GLU396, and VAL397 of BRD-3 BD2; and PRO375, GLU438,and VAL439 of BRD-4 BD2 and 3) testing the ability of the interactingcompound to inhibit the binding of the second bromodomain to afluorescent ligand in a fluorescent anisotropy binding assay; whereinthe fluorescent ligand is a compound having the structure

 conjugated with a fluorescent label at the terminal amino group (—NH₂)of the compound; and wherein the interacting compound which is able toinhibit the binding of the second bromodomain to the fluorescent ligandin the fluorescent anisotropy binding assay is identified as thecompound which inhibits the binding of the second bromodomain of each ofhuman BRD-2, BRD-3 and BRD-4 to the acetylated lysine residues of theirphysiological partner proteins.
 2. A process for the identification of acompound which inhibits the binding of the second bromodomain of each ofhuman BRD-2, BRD-3, and BRD-4 to acetylated lysine residues of theirphysiological partner proteins which process comprises the steps of: 1)selecting compounds with a molecular weight in the range 100 to 750 Da;2) determining which of those selected compounds is an interactingcompound, wherein an interacting compound will: a) form a hydrogenbonding interaction in which the compound accepts a hydrogen bond fromthe sidechain NH₂ group of the asparagine residue found at: ASN429 ofBRD-2 BD2, or ASN391 of BRD-3 BD2, or ASN433 of BRD-4 BD2 and b) accepta water-mediated hydrogen bond in which the compound accepts a hydrogenbond from a water that is itself hydrogen-bonded to the sidechainhydroxyl of the tyrosine residue found at the TYR386 of BRD-2 BD2, orTYR348 of BRD-3 BD2, or TYR390 of BRD-4 BD2 and c) form a Van der Waalsinteraction with a lipophilic binding region of a binding pocket suchthat one or more heavy atoms of the said compounds lie within a 7.5 Årange of at least one heavy atom of each of the 3 residues of the secondbromodomain wherein said second bromodomain residues are selected fromthe group consisting of: PRO371, ASP434, and VAL435 of BRD-2 BD2;PRO333, GLU396, and VAL397 of BRD-3 BD2; and PRO375, GLU438, and VAL439of BRD-4 BD2; and 3) testing the ability of the interacting compound toinhibit the binding of the second bromodomain to a fluorescent ligand ina fluorescent anisotropy binding assay; wherein the fluorescent ligandis a compound having the structure

 conjugated with a fluorescent label at the terminal amino group (—NH₂)of the compound; and wherein the interacting compound which is able toinhibit the binding of the second bromodomain to the fluorescent ligandin the fluorescent anisotropy binding assay is identified as thecompound which inhibits the binding of the second bromodomain of each ofhuman BRD-2, BRD-3 and BRD-4 to the acetylated lysine residues of theirphysiological partner proteins.
 3. A process according to claim 1wherein said molecular weight is in the range 100 to 500 Da.
 4. Aprocess according to claim 2 wherein said molecular weight is in therange 100 to 500 Da.
 5. A process for the identification of a compoundwhich inhibits the binding of the second bromodomain of each of humanBRD-2, BRD-3, and BRD-4 to acetylated lysine residues of theirphysiological partner proteins which process comprises the steps of: 1)selecting compounds with a molecular weight in the range 100 to 750 Da;and 2) determining which of those selected compounds is an interactingcompound, wherein an interacting compound will: a) form a hydrogenbonding interaction in which the compound accepts a hydrogen bond fromthe sidechain NH₂ group of the asparagine residue found at: ASN429 ofBRD-2 BD2, or ASN391 of BRD-3 BD2, or ASN433 of BRD-4 BD2 and b) form aVan der Waals interaction with a lipophilic binding region of a bindingpocket such that one or more heavy atoms of the said compounds liewithin a 7.5 Å range of at least one heavy atom of each of the 3residues of the second bromodomain wherein said bromodomain residues areselected from the group consisting of: PRO371, ASP434, and VAL435 ofBRD-2 BD2; PRO333, GLU396, and VAL397 of BRD-3 BD2; and PRO375, GLU438,and VAL439 of BRD-4 BD2 and 3) testing the ability of the interactingcompound to inhibit the binding of the second bromodomain to afluorescent ligand in a fluorescent anisotropy binding assay, whereinthe fluorescent ligand is a compound having the structure

 conjugated with a fluorescent label at the terminal amino group (—NH₂)of the compound, wherein the second bromodomain is comprised in a humanbromodomain protein that comprises a histidine tag; and wherein theinteracting compound which is able to inhibit the binding of the secondbromodomain to the fluorescent ligand in the fluorescent anisotropybinding assay is identified as the compound which inhibits the bindingof the second bromodomain of each of human BRD-2, BRD-3 and BRD-4 to theacetylated lysine residues of their physiological partner proteins.
 6. Aprocess according to claim 5 wherein said molecular weight is in therange 100 to 500 Da.
 7. A process for the identification of a compoundwhich inhibits the binding of the second bromodomain of each of humanBRD-2, BRD-3, and BRD-4 to acetylated lysine residues of theirphysiological partner proteins which process comprises the steps of: 1)selecting compounds with a molecular weight in the range 100 to 750 Da;2) determining which of those selected compounds is an interactingcompound, wherein an interacting compound will: a) form a hydrogenbonding interaction in which the compound accepts a hydrogen bond fromthe sidechain NH2 group of the asparagine residue found at: ASN429 ofBRD-2 BD2, or ASN391 of BRD-3 BD2, or ASN433 of BRD-4 BD2 and b) accepta water-mediated hydrogen bond in which the compound accepts a hydrogenbond from a water that is itself hydrogen-bonded to the sidechainhydroxyl of the tyrosine residue found at the TYR386 of BRD-2 BD2, orTYR348 of BRD-3 BD2, or TYR390 of BRD-4 BD2 and c) form a Van der Waalsinteraction with a lipophilic binding region of a binding pocket suchthat one or more heavy atoms of the said compounds lie within a 7.5 Årange of at least one heavy atom of each of the 3 residues of the secondbromodomain wherein said second bromodomain residues are selected fromthe group consisting of: PRO371, ASP434, and VAL435 of BRD-2 BD2;PRO333, GLU396, and VAL397 of BRD-3 BD2; and PRO375, GLU438, and VAL439of BRD-4 BD2; and 3) testing the ability of the interacting compound toinhibit the binding of the second bromodomain to a fluorescent ligand ina fluorescent anisotropy binding assay, wherein the fluorescent ligandis a compound having the structure

 conjugated with a fluorescent label at the terminal amino group (—NH₂)of the compound, wherein the second bromodomain is comprised in a humanbromodomain protein that comprises a histidine tag; and wherein theinteracting compound which is able to inhibit the binding of the secondbromodomain to the fluorescent ligand in the fluorescent anisotropybinding assay is identified as the compound which inhibits the bindingof the second bromodomain of each of human BRD-2, BRD-3 and BRD-4 to theacetylated lysing residues of their physiological partner proteins.
 8. Aprocess according to claim 7 wherein said molecular weight is in therange 100 to 500 Da.